Science.gov

Sample records for delta endotoxins cry1ac

  1. Disruption of Ha_BtR alters binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to midgut BBMVs of Helicoverpa armigera.

    PubMed

    Xu, Xinjun; Wu, Yidong

    2008-01-01

    Disruption of the Ha_BtR (a cadherin gene) is genetically linked to resistance to Cry1Ac delta-endotoxin of Bacillus thuringiensis in the GYBT strain of Helicoverpa armigera. Brush border membrane vesicles (BBMVs) prepared from midguts of both the Cry1Ac-resistant GYBT strain (homozygous for a deletion knockout of Ha_BtR) and the susceptible GY strain (homozygous for the wild type of Ha_BtR) possessed saturable and specific binding ability to (125)I-Cry1Ac. The binding constant (K(d)) of the GY strain was significantly lower than that of the resistant GYBT strain, whereas their binding site concentrations (B(max)) were similar. When midgut BBMVs were reacted directly with streptavidin conjugated to horseradish peroxidase, the GY strain had very clear 120- and 85-kDa protein bands, which indicated that the 120- and 85-kDa bands are endogenous biotin-containing proteins. However, the GYBT strain almost completely lost these two biotin-containing proteins. Ligand blotting with biotinylated Cry1Ac toxin showed midgut BBMVs of the GY strain contain five protein bands of 210-, 190-, 150-, 120-, and 85-kDa, respectively, while BBMVs of the GYBT strain contain only two protein bands of 150- and 120-kDa. 120-kDa bands may consist of two proteins with coincidentally the same molecular weight (putatively, an APN and a biotin-containing protein). Our results showed that the binding pattern of Cry1Ac to midgut BBMVs of H. armigera was altered quantitatively and qualitatively by knockout of Ha_BtR. There are multiple Cry1Ac-binding proteins in the midgut of susceptible H. armigera, but only the Ha_BtR can be considered as a putative functional receptor of Cry1Ac. Possible involvement of other receptor proteins in the intoxication process in vivo could not be excluded. PMID:17681529

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

    PubMed

    Akhurst, Raymond Joseph; James, William; Bird, Lisa Jane; Beard, Cheryl

    2003-08-01

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

  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. 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. PMID:21654089

  5. 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

  6. 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.

  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. PMID:25195523

  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. Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

    PubMed

    Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

    2004-04-01

    The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. PMID:15027071

  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. Cry1Ac toxicity enhancement towards lepidopteran pest Ephestia kuehniella through its protection against excessive proteolysis.

    PubMed

    Elleuch, Jihen; Jaoua, Samir; Tounsi, Slim; Zghal, Raida Z

    2016-09-15

    Bacillus thuringiensis has been extensively used in agroecosystems for four decades due to its high specific toxicity. Strategies based on B. thuringiensis proteins combinations for the improvement of its activity present an important focus for biopesticides development. However, the widespread use of B. thuringiensis δ-endotoxins has often been challenged by a lack of understanding of the target insect physiology as well as its midgut biochemistry. In the present investigation, we have evidenced and explained the toxicity improvement of Cry1Ac δ-endotoxins against Ephestia kuehniella larvae through in vivo combination with P20 helper protein. Tracking the fate of Cry1Ac in tested midgut larvae showed considerable differences between δ-endotoxins produced in the presence of P20 and those produced in its absence which could explain the obtained larvicidal activity enhancement. The P20 presence slightly increased Cry1Ac inclusions solubility in E. kuehniella midgut conditions. However, a protection against excessive degradation of protoxin and toxin forms of Cry1Ac was strongly decreased in the case of δ-endotoxins produced in the presence of P20 as compared to those from P20 lacking control. Thus, the P20 protective effect on Cry1Ac after larvae ingestion has been proven. This finding could be helpful to further understand the roles of P20 helper protein in toxicity enhancement of B. thuringiensis toxins. PMID:27452929

  12. Susceptibility of legume pod borer (LPB), Maruca vitrata to delta-endotoxins of Bacillus thuringiensis (Bt) in Taiwan.

    PubMed

    Srinivasan, R

    2008-01-01

    Baseline susceptibility of legume pod borer (LPB) to the insecticidal crystal proteins (ICPs) from Bacillus thuringiensis, viz, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca and Cry2Aa was assessed in Taiwan. Insect bioassays were performed by incorporating the Bt delta-endotoxins into the LPB artificial diet. The efficacy of different Bt delta-endotoxins against second instar larvae of LPB showed that the toxin Cry1Ab was the most potent toxin (LC(50) 0.207ppm), followed by Cry1Ca, Cry1Aa, Cry2Aa and Cry1Ac in descending order, with LC(50)s 0.477ppm, 0.812ppm, 1.058ppm and 1.666ppm, respectively. Hence, Cry1Ab and/or Cry1Ca toxins would provide effective control of early larval stages of LPB.

  13. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Acquisition of Cry1Ac protein by non-target arthropods in Bt soybean fields.

    PubMed

    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.

  16. 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

  17. 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. PMID:25843583

  18. Influences of Cry1Ac Broccoli on Larval Survival and Oviposition of Diamondback Moth

    PubMed Central

    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. PMID:25843583

  19. 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.

  20. Baseline susceptibility to Cry1Ac insecticidal protein in Heliothis virescens (Lepidoptera: Noctuidae) populations in Brazil.

    PubMed

    Albernaz, K C; Merlin, B L; Martinelli, S; Head, G P; Omoto, C

    2013-08-01

    The tobacco budworm, Heliothis virescens (F.), is one of the target pests of genetically modified cotton expressing Cry1Ac insecticidal protein (Bt cotton) derived from Bacillus thuringiensis Berliner. This study was conducted to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac to establish a baseline for use in an insect resistance management program for Bt cotton in Brazil. Insects were sampled from the main Brazilian cotton-growing regions (Bahia, Mato Grosso, Mato Grosso do Sul, and Goiás) during the cropping seasons of 2007/08 and 2008/09. Cry1Ac susceptibility was estimated by using diet incorporation bioassays. H. virescens was highly susceptible to Cry1Ac protein. The estimated LC50 values varied from 0.18 to 0.66 microg of Cry1Ac/ml of diet among the 2007-2008 populations (approximately 3.7-fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 microg of Cry1Ac/ml of diet for the 2007-2008 populations (approximately 3.0-fold variation). A joint analysis of the mortality data across all tested populations was used to develop and validate the diagnostic concentrations of 3.1 and 5.6 microg of Cry1Ac/ml of diet, the upper bound of the confidence interval and twice the LC99 were selected, for resistance monitoring programs of H. virescens to Cry1Ac protein in Brazil.

  1. 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

  2. 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. PMID:27257885

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

    PubMed Central

    Ojha, Abhishek; Sree, K Sowjanya; Sachdev, Bindiya; Rashmi, MA; Ravi, KC; Suresh, PJ; 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. PMID:25523173

  4. 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

  5. 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

  6. Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen.

    PubMed

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

    2012-08-01

    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

  7. Identification of an alkaline phosphatase as a putative Cry1Ac binding protein in Ostrinia furnacalis (Guenée).

    PubMed

    Jin, Tingting; Duan, Xiaoli; Bravo, Alejandra; Soberón, Mario; Wang, Zhenying; He, Kanglai

    2016-07-01

    Asian corn borer (ACB), Ostrinia furnacalis, is an important insect pest of maize susceptible to different Cry1A toxins. Based on amino acid sequence alignment of ALP sequences from lepidopteran larvae an alp gene was cloned from ACB, named ofalp. Pull dawn assays using biotinylated Cry1Ac and brush border membrane vesicles isolated from second instar ACB larvae showed that four proteins of 50, 65, 68 and 70kDa precipitated with the Cry1Ac. The 65kDa band cross-reacted with the anti-OfALP monoclonal antibody. GalNac was able to release the binding of Cry1Ac to the 65kDa OfALP in pull down assays. A 37kDa fragment from residues D173 to D473 of OfALP was cloned and expressed in Escherichia coli cells. We show that this ALP-fragment was able to bind Cry1Ac in ligand blot analysis. Our data also indicate that different ALP isoforms or variants may be also Cry1Ac binding proteins since more ALP enzymatic activity was pull down with Cry1Ac than with anti-OfALP antibody. We also analyzed the expression levels of ALP throughout the larval development by qPCR and ALP enzymatic activity. Our data indicated that ALP expression in ACB was observed preferentially in young instar larvae. Finally, we show that resistance in O. furnacalis ACB-AcR strain resistant to Cry1Ac did not correlate with changes in expression of this ALP protein since it shows similar gene expression of ofalp than the susceptible insect strain. Identification of Cry1Ac receptors will help to understand mechanism of action of Cry1Ac in O. furnacalis and to understand mechanism of Cry toxin resistance. Our data indicate that at least one ALP protein is involved in the binding interaction with Cry1Ac in O. furnacalis. PMID:27265829

  8. Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Zhang, Shaoping; Cheng, Hongmei; Gao, Yulin; Wang, Guirong; Liang, Gemei; Wu, Kongming

    2009-07-01

    A Cry1Ac-resistant strain (Bt-R) of Helicoverpa armigera, with 2971-fold resistance, was derived by selection with Cry1Ac toxin for 75 generations. We used cDNA-amplified fragment length polymorphism analysis to identify those genes differentially expressed in the Cry1Ac-resistant and -susceptible strains, which revealed 212 differentially expressed transcripts among 2000 screened cDNAs. Among these transcript-derived fragments (TDFs), 37 showed some homology to known sequences, including Aminopeptidase N (APN), which is expressed in the midgut epithelium and has been implicated as a Cry1A subfamily receptor in several moths, including H. armigera. We confirmed the TDF by RT-PCR and identified a deletion mutation of apn1 in the Bt-R strain. We expressed the TDF in bacteria. The partial HaAPN1-96S wild-type protein, bound to Cry1Ac on ligand blots, whereas HaAPN1-BtR did not. This suggested that HaAPN1 is a receptor for Bt Cry1Ac and that its deletion mutation is associated with Cry1Ac resistance in H. armigera. The absence of one binding site is responsible for its resistance to Cry1Ac. We developed an allele-specific PCR to monitor whether the apn1 gene in an H. armigera field population produced a similar mutation. No deleted mutants were found in 2250 individuals collected from the field in 2006-2007. PMID:19376227

  9. 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...

  10. 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...

  11. 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...

  12. 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. PMID:24484070

  13. 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.

  14. 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

  15. Field Evolved Resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis Toxin Cry1Ac in Pakistan

    PubMed Central

    Alvi, Anwaar H. K.; Sayyed, Ali H.; Naeem, Muhammad; Ali, Muhammad

    2012-01-01

    Helicoverpa armigera (Hübner) is one of the most destructive pests of several field and vegetable crops, with indiscriminate use of insecticides contributing to multiple instances of resistance. In the present study we assessed whether H. armigera had developed resistance to Bt cotton and compared the results with several conventional insecticides. Furthermore, the genetics of resistance was also investigated to determine the inheritance to Cry1Ac resistance. To investigate the development of resistance to Bt cotton, and selected foliar insecticides, H. armigera populations were sampled in 2010 and 2011 in several cotton production regions in Pakistan. The resistance ratios (RR) for Cry1Ac, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin and deltamethrin were 580-fold, 320-, 1110-, 1950-, 200-, 380, 690, and 40-fold, respectively, compared with the laboratory susceptible (Lab-PK) population. Selection of the field collected population with Cry1Ac in 2010 for five generations increased RR to 5440-fold. The selection also increased RR for deltamethrin, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin to 125-folds, 650-, 2840-, 9830-, 370-, 3090-, 1330-fold. The estimated LC50s for reciprocal crosses were 105 µg/ml (Cry1Ac-SEL female × Lab-PK male) and 81 g µg/ml (Lab-PK female × Cry1Ac-SEL male) suggesting that the resistance to Cry1Ac was autosomal; the degree of dominance (DLC) was 0.60 and 0.57 respectively. Mixing of enzyme inhibitors significantly decreased resistance to Cry1Ac suggesting that the resistance to Cry1Ac and other insecticides tested in the present study was primarily metabolic. Resistance to Cry1Ac was probably due to a single but unstable factor suggesting that crop rotation with non-Bt cotton or other crops could reduce the selection pressure for H. armigera and improve the sustainability of Bt cotton. PMID:23077589

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

    PubMed

    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

  17. 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

  18. 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. PMID:23470135

  19. 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

  20. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

    PubMed

    Chen, Wenbo; Liu, Chenxi; Xiao, Yutao; Zhang, Dandan; Zhang, Yongdong; Li, Xianchun; Tabashnik, Bruce E; Wu, Kongming

    2015-01-01

    Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects.

  1. 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

  2. Distribution and Metabolism of Bt-Cry1Ac Toxin in Tissues and Organs of the Cotton Bollworm, Helicoverpa armigera

    PubMed Central

    Zhao, Zhuoya; Li, Yunhe; Xiao, Yutao; Ali, Abid; Dhiloo, Khalid Hussain; Chen, Wenbo; Wu, Kongming

    2016-01-01

    Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have been widely used in transgenic crops due to their toxicity against insect pests. However, the distribution and metabolism of these toxins in insect tissues and organs have remained obscure because the target insects do not ingest much toxin. In this study, several Cry1Ac-resistant strains of Helicoverpa armigera, fed artificial diets containing high doses of Cry1Ac toxin, were used to investigate the distribution and metabolism of Cry1Ac in their bodies. Cry1Ac was only detected in larvae, not in pupae or adults. Also, Cry1Ac passed through the midgut into other tissues, such as the hemolymph and fat body, but did not reach the larval integument. Metabolic tests revealed that Cry1Ac degraded most rapidly in the fat body, followed by the hemolymph, peritrophic membrane and its contents. The toxin was metabolized slowly in the midgut, but was degraded in all locations within 48 h. These findings will improve understanding of the functional mechanism of Bt toxins in target insects and the biotransfer and the bioaccumulation of Bt toxins in arthropod food webs in the Bt crop ecosystem. PMID:27399776

  3. A Toxin-Binding Alkaline Phosphatase Fragment Synergizes Bt Toxin Cry1Ac against Susceptible and Resistant Helicoverpa armigera

    PubMed Central

    Xiao, Yutao; Zhang, Dandan; Zhang, Yongdong; Li, Xianchun; Tabashnik, Bruce E.; Wu, Kongming

    2015-01-01

    Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects. PMID:25885820

  4. Distribution and Metabolism of Bt-Cry1Ac Toxin in Tissues and Organs of the Cotton Bollworm, Helicoverpa armigera.

    PubMed

    Zhao, Zhuoya; Li, Yunhe; Xiao, Yutao; Ali, Abid; Dhiloo, Khalid Hussain; Chen, Wenbo; Wu, Kongming

    2016-01-01

    Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have been widely used in transgenic crops due to their toxicity against insect pests. However, the distribution and metabolism of these toxins in insect tissues and organs have remained obscure because the target insects do not ingest much toxin. In this study, several Cry1Ac-resistant strains of Helicoverpa armigera, fed artificial diets containing high doses of Cry1Ac toxin, were used to investigate the distribution and metabolism of Cry1Ac in their bodies. Cry1Ac was only detected in larvae, not in pupae or adults. Also, Cry1Ac passed through the midgut into other tissues, such as the hemolymph and fat body, but did not reach the larval integument. Metabolic tests revealed that Cry1Ac degraded most rapidly in the fat body, followed by the hemolymph, peritrophic membrane and its contents. The toxin was metabolized slowly in the midgut, but was degraded in all locations within 48 h. These findings will improve understanding of the functional mechanism of Bt toxins in target insects and the biotransfer and the bioaccumulation of Bt toxins in arthropod food webs in the Bt crop ecosystem. PMID:27399776

  5. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera.

    PubMed

    Chen, Wenbo; Liu, Chenxi; Xiao, Yutao; Zhang, Dandan; Zhang, Yongdong; Li, Xianchun; Tabashnik, Bruce E; Wu, Kongming

    2015-01-01

    Evolution of resistance by insects threatens the continued success of pest control using insecticidal crystal (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) in sprays and transgenic plants. In this study, laboratory selection with Cry1Ac yielded five strains of cotton bollworm, Helicoverpa armigera, with resistance ratios at the median lethal concentration (LC50) of activated Cry1Ac ranging from 22 to 1700. Reduced activity and reduced transcription of an alkaline phosphatase protein that binds Cry1Ac was associated with resistance to Cry1Ac in the four most resistant strains. A Cry1Ac-binding fragment of alkaline phosphatase from H. armigera (HaALP1f) was not toxic by itself, but it increased mortality caused by Cry1Ac in a susceptible strain and in all five resistant strains. Although synergism of Bt toxins against susceptible insects by toxin-binding fragments of cadherin and aminopeptidase N has been reported previously, the results here provide the first evidence of synergism of a Bt toxin by a toxin-binding fragment of alkaline phosphatase. The results here also provide the first evidence of synergism of a Bt toxin by any toxin-binding peptide against resistant insects. PMID:25885820

  6. Distribution and Metabolism of Bt-Cry1Ac Toxin in Tissues and Organs of the Cotton Bollworm, Helicoverpa armigera.

    PubMed

    Zhao, Zhuoya; Li, Yunhe; Xiao, Yutao; Ali, Abid; Dhiloo, Khalid Hussain; Chen, Wenbo; Wu, Kongming

    2016-01-01

    Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have been widely used in transgenic crops due to their toxicity against insect pests. However, the distribution and metabolism of these toxins in insect tissues and organs have remained obscure because the target insects do not ingest much toxin. In this study, several Cry1Ac-resistant strains of Helicoverpa armigera, fed artificial diets containing high doses of Cry1Ac toxin, were used to investigate the distribution and metabolism of Cry1Ac in their bodies. Cry1Ac was only detected in larvae, not in pupae or adults. Also, Cry1Ac passed through the midgut into other tissues, such as the hemolymph and fat body, but did not reach the larval integument. Metabolic tests revealed that Cry1Ac degraded most rapidly in the fat body, followed by the hemolymph, peritrophic membrane and its contents. The toxin was metabolized slowly in the midgut, but was degraded in all locations within 48 h. These findings will improve understanding of the functional mechanism of Bt toxins in target insects and the biotransfer and the bioaccumulation of Bt toxins in arthropod food webs in the Bt crop ecosystem.

  7. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

    PubMed

    Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

    2016-09-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. PMID:27521921

  8. Long-term toxicity study on transgenic rice with Cry1Ac and sck genes.

    PubMed

    Zhang, Min; Zhuo, Qin; Tian, Yuan; Piao, Jianhua; Yang, Xiaoguang

    2014-01-01

    In the present work, we evaluated the chronic effects of the transgenic insect-resistant rice carrying Cry1Ac and sck genes on Sprague-Dawley (SD) rats through a 78-week feeding study. Based on the gender and weight, 180 SD rats were randomly and evenly assigned into three groups. GM rice and non-GM rice were separately formulated into diets at high levels. AIN-93 diet was used as a nutritional control. Body weight, food consumption, hematology and serum chemistry were monitored regularly. Rats were sacrificed for organ weight measurement and pathological examination at 52 weeks and 78 weeks. Body weight, food consumption, mortality rates, tumor incidences and pathological findings showed no significant difference among the three groups. Although certain differences in some hematology, serum chemistry parameters and relative organ weights were observed between GM rice group and control groups, they were not considered as treatment-related. Taken together, long-term intake of transgenic rice carrying Cry1Ac and sck genes at a high level exerts no unintended adverse effects on rats.

  9. Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin

    PubMed Central

    Li, Min; Zhu, Min; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-01-01

    Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL−1 and a working range 0.010–1.0 μg·mL−1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system. PMID:25474492

  10. 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

  11. Uniform orientation of biotinylated nanobody as an affinity binder for detection of Bacillus thuringiensis (Bt) Cry1Ac toxin.

    PubMed

    Li, Min; Zhu, Min; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-01-01

    Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL-1 and a working range 0.010-1.0 μg·mL-1. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system. PMID:25474492

  12. 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

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

    PubMed Central

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

    2009-01-01

    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. PMID:19581574

  14. 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

  15. 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.

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

    PubMed

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

    2015-01-01

    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. PMID:26345776

  17. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fat body is one of the most abundant not-gut hemocoelic tissues that may serve as the potential target tissues of Cry toxins in insect larvae, but nothing is known about its susceptibility to any Cry toxins. To estimate the susceptibilities to Cry1Ac and Cry2Ab of fat body, we measured the cytotoxi...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Electrogenerated chemiluminescence immunosensor for Bacillus thuringiensis Cry1Ac based on Fe3O4@Au nanoparticles.

    PubMed

    Li, Jianping; Xu, Qian; Wei, Xiaoping; Hao, Zaibin

    2013-02-20

    A highly sensitive electrochemiluminescence (ECL) immunosensor for Cry1Ac was fabricated. The primary antibody anti-Cry1Ac was immobilized onto core-shell structural Fe(3)O(4)@Au nanoparticles. The antigen and glucose-oxidase-labeled secondary antibody were then successively combined to form sandwich-type immunocomplexes through a specific interaction. The magnetic particles loaded with sandwich immune complexes were attracted to a magnet-controlled glass carbon electrode (GCE) by an external magnet applied on top of the GCE. ECL was generated by the reaction between luminol and hydrogen peroxide derived from the enzymatic reaction in the presence of glucose. The sensors exhibited high sensitivity and a wide linear range for Bacillus thuringiensis Cry1Ac detection from 0 to 6 ng/mL, as well as a detection limit of 0.25 pg/mL (S/N = 3). The sensor is one of the most sensitive sensors for Cry1Ac, which can be easily renewed and conveniently used. PMID:23317307

  2. Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae.

    PubMed

    Jurat-Fuentes, Juan L; Adang, Michael J

    2004-08-01

    We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [125I]Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection, reduced soybean agglutinin binding to HvALP from Cry1Ac resistant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible (YDK and F1 generation from backcrosses) and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae. PMID:15265032

  3. 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

  4. 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... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of...

  5. 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... PLANT-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.510 Bacillus thuringiensis Cry1Ac protein in all plants; exemption from the requirement of a tolerance. Residues of...

  6. Impact of cry1AC-carrying Brassica rapa subsp. pekinensis on leaf bacterial community.

    PubMed

    Kim, Young Tae; Lee, Kang Seon; Kim, Moon Jung; Kim, Seung Bum

    2009-02-01

    The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.

  7. Detection and characterization of cry1Ac transgene construct in Bt cotton: multiple polymerase chain reaction approach.

    PubMed

    Singh, Chandra K; Ojha, Abhishek; Kachru, Devendra N

    2007-01-01

    To comply with international labeling regulations for genetically modified (GM) crops and food, and to enable proper identification of GM organisms (GMOs), effective methodologies and reliable approaches are needed. The spurious and unapproved GM planting has contributed to crop failures and commercial losses. To ensure effective and genuine GM cultivation, a methodology is needed to detect and identify the trait of interest and concurrently evaluate the structural and functional stability of the transgene insert. A multiple polymerase chain reaction (PCR) approach was developed for detection, identification, and gene stability confirmation of cry1Ac transgene construct in Bt cotton. As many as 9 samples of Bt cotton hybrid seeds comprising 3 approved Bt hybrids, MECH-12Bt, MECH-162Bt, MECH-184Bt, and a batch of 6 nonapproved Bt hybrids were tested. Initially, single standard PCR assays were run to amplify predominant GM DNA sequences (CaMV 35S promoter, nos terminator, and npt-II marker gene); a housekeeping gene, Gossypium hirsutum fiber-specific acyl carrier protein gene (acp1); a trait-specific transgene (cry1Ac); and a sequence of 7S 3' transcription terminator which specifically borders with 3' region of cry1Ac transgene cassette. The concurrent amplification of all sequences of the entire cassette was performed by 3 assays, duplex, triplex, and quadruplex multiplex PCR assays, under common assay conditions. The identity of amplicons was reconfirmed by restriction endonuclease digestion profile. The 2 distinct transgene cassettes, cry1Ac and npt-II, of the Bt cotton were amplified using the respective forward primer of promoter and reverse primer of terminator. The resultant amplicons were excised, eluted, and purified. The purified amplicons served as template for nested PCR assays. The nested PCR runs confirmed the transgene construct orientation and identity. The limit of detection as established by our assay for GM trait (cry1Ac) was 0.1%. This approach

  8. Genetic and Biochemical Approach for Characterization of Resistance to Bacillus thuringiensis Toxin Cry1Ac in a Field Population of the Diamondback Moth, Plutella xylostella

    PubMed Central

    Sayyed, Ali H.; Haward, Robert; Herrero, Salvador; Ferré, Juan; Wright, Denis J.

    2000-01-01

    Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F4 to F8) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F9 found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F9. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F9 to F13) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with 125I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F15). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F2 progeny from a backcross of F1 progeny with ROTH was tested with a concentration of Cry1Ac which would kill

  9. 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.

  10. 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.

  11. 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. PMID:25890087

  12. 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. PMID:25721170

  13. Impact of Water Content and Temperature on the Degradation of Cry1Ac Protein in Leaves and Buds of Bt Cotton in the Soil

    PubMed Central

    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. PMID:25559638

  14. Cry1Ac toxin induces macrophage activation via ERK1/2, JNK and p38 mitogen-activated protein kinases.

    PubMed

    Torres-Martínez, Marilu; Rubio-Infante, Néstor; García-Hernández, Ana Lilia; Nava-Acosta, Raúl; Ilhuicatzi-Alvarado, Damaris; Moreno-Fierros, Leticia

    2016-09-01

    The Cry1Ac toxin from Bacillus thuringiensis is used commercially as a bio-insecticide and is expressed in transgenic plants that are used for human and animal consumption. Although it was originally considered innocuous for mammals, the Cry1Ac toxin is not inert and has the ability to induce mucosal and systemic immunogenicity. Herein, we examined whether the Cry1Ac toxin promotes macrophage activation and explored the signalling pathways that may mediate this effect. Treatment of primary and RAW264.7 macrophages with the Cry1Ac toxin resulted in upregulation of the costimulatory molecules CD80, CD86 and ICOS-L and enhanced production of nitric oxide, the chemokine MCP-1 and the proinflammatory cytokines TNF-α and IL-6. Remarkably, the Cry1Ac toxin induced phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK1/2, JNK and p38 and promoted nuclear translocation of nuclear factor-kappa B (NF-κB) p50 and p65. p38 and ERK1/2 MAPKs were involved in this effect, as indicated by the Cry1Ac-induced upregulation of CD80 and IL-6 and TNF-α abrogation by the p38 MAPK inhibitor SB203580. Furthermore, treatment the MEK1/2 kinase inhibitor PD98059 blocked increases in MCP-1 secretion and augmented Cry1Ac-induced ICOS-L upregulation. These data demonstrate the capacity of the Cry1Ac toxin to induce macrophage activation via the MAPK and NF-κB pathways. PMID:27394658

  15. 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.

  16. The midgut cadherin-like gene is not associated with resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella (L.).

    PubMed

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

    2015-03-01

    The Gram-positive bacterium Bacillus thuringiensis (Bt) produces Cry toxins that have been used to control important agricultural pests. Evolution of resistance in target pests threatens the effectiveness of these toxins when used either in sprayed biopesticides or in Bt transgenic crops. Although alterations of the midgut cadherin-like receptor can lead to Bt Cry toxin resistance in many insects, whether the cadherin gene is involved in Cry1Ac resistance of Plutella xylostella (L.) remains unclear. Here, we present experimental evidence that resistance to Cry1Ac or Bt var. kurstaki (Btk) in P. xylostella is not due to alterations of the cadherin gene. The bona fide P. xylostella cadherin cDNA sequence was cloned and analyzed, and comparisons of the cadherin cDNA sequence among susceptible and resistant P. xylostella strains confirmed that Cry1Ac resistance was independent of mutations in this gene. In addition, real-time quantitative PCR (qPCR) indicated that cadherin transcript levels did not significantly differ among susceptible and resistant P. xylostella strains. RNA interference (RNAi)-mediated suppression of cadherin gene expression did not affect larval susceptibility to Cry1Ac toxin. Furthermore, genetic linkage assays using four cadherin gDNA allelic biomarkers confirmed that the cadherin gene is not linked to resistance against Cry1Ac in P. xylostella. Taken together, our findings demonstrate that Cry1Ac resistance of P. xylostella is independent of the cadherin gene. PMID:25595643

  17. 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. PMID:25437213

  18. 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

  19. 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...

  20. 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...

  1. 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...

  2. 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.

  3. Inheritance Patterns, Dominance and Cross-Resistance of Cry1Ab- and Cry1Ac-Selected Ostrinia furnacalis (Guenée)

    PubMed Central

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

    2014-01-01

    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. PMID:25216083

  4. 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

    2014-09-01

    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. PMID:25216083

  5. Cadherin mutation linked to resistance to Cry1Ac affects male paternity and sperm competition in Helicoverpa armigera

    PubMed Central

    Zhang, Haonan; Du, Bing; Higginson, Dawn M.; Carrière, Yves; Wu, Yidong

    2015-01-01

    Several lepidopteran pests of cotton have cadherin-based resistance to the Bacillus thuringiensis (Bt) toxin Cry1Ac. Cadherins are transmembrane proteins that mediate cell-cell adhesion and tissue morphogenesis, suggesting that fitness costs associated with cadherin mutations may be present in many aspects of life history. To evaluate whether cadherin-based resistance is associated with fitness costs reducing male paternity in Helicoverpa armigera, we examined the effects of a major cadherin resistance allele on sperm competition within and between male ejaculates. When homozygous resistant and susceptible males competed for fertilization of a homozygous resistant or susceptible female, fertilization success was high in males with a different cadherin genotype than females and low in males with the same cadherin genotype as females. Single matings between heterozygous males and susceptible females produced offspring within typical Mendelian ratios. Heterozygous males mated to resistant females, however, resulted in a disproportionate number of heterozygous offspring. While these results show that cadherin-based resistance to Cry1Ac has significant impacts on paternity in H. armigera, there was no evidence that costs associated with resistance consistently reduced male paternity. Rather, effects of cadherin-based resistance on paternity depended on interactions between male and female genotypes and differed when males or sperm competed for fertilization of females, which complicates assessment of impacts of cadherin resistance alleles on resistance evolution. PMID:25220924

  6. Changes in Cry1Ac Bt transgenic cotton in response to two environmental factors: temperature and insect damage.

    PubMed

    Olsen, K M; Daly, J C; Finnegan, E J; Mahon, R J

    2005-08-01

    The efficacy of Cry1Ac Bacillus thuringiensis (Bt) cotton plants against field populations of Helicoverpa armigera (Hübner) has been inconsistent over the growing season. Any reduction in efficacy (where efficacy is the capacity of the plant to affect the survival of the insect) increases the opportunities for H. armigera to evolve resistance to Bt toxin. Changes in efficacy could be due to changes at the level of gene expression and/or in the physiological makeup of the plant and may be induced by environmental conditions. Two environmental factors, temperature and insect damage, were investigated. Temperature was found to affect efficacy, whether plants were grown at different temperatures continuously or were exposed to a change in temperature for a short period. Damage caused by chewing insects (H. armigera larvae) produced a dramatic increase in the efficacy of presquare Bt cotton. In contrast, damage by sucking insects (aphids) did not induce changes in efficacy. Changes in efficacy seemed to be mediated through modification of the physiological background of the plant rather than changes in the level of Cry1Ac expression or in the concentration of the Bt toxin. The impact of the non-Bt responses of plants on strains of H. armigera should be evaluated. It is possible that by enhancing existing defensive mechanisms of plants, the rate of evolution of resistance to Bt toxins could be retarded by increasing the plants overall toxicity through the additive effects of the toxins and plant defenses.

  7. [Comparison between transgenic insect-resistant cotton expressing Cry1Ac protein and its parental variety in rhizospheric fungal diversity].

    PubMed

    Pan, Jian-Gang; Jiao, Hai-Hua; Bai, Zhi-Hui; Qi, Hong-Yan; Ma, An-Zhou; Zhuang, Guo-qiang; Zhang, Hong-xun

    2014-11-01

    The dynamics of rhizospheric fungal diversity and biomass at different sampling stages associated with two transgenic insectresistant cottons expressing Cry1Ac protein and their control varieties were studied under greenhouse conditions, followed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time polymerase chain reaction (Q-PCR), in order to evaluate the ecological security of planting transgenic cotton expressing Cry1Ac protein. The results indicated that the fungal superior bands in rhizosphere of transgenic Bt cotton were similar with that of control cotton at four sampling stages, the more obvious difference in the blurred bands among transgenic Bt cotton, JM20 and SHIYUAN321 was detected. The rhizospheric fungal biomass of transgenic Bt cotton SGK321 was significantly lower than that of its parental control cotton at seedling stage, while the slight decrease in fungal biomass of transgenic Bt cotton XP188 was detected at boll forming stage, the ill-defined decrease, even growing tendency in two transgenic Bt cottons was detected at other stages. However, the difference of rhizospheric fungal community compositions and biomass was not only existed between transgenic cotton and its control, but also between SHIYUAN321 and JM20, and the same phenomenon was also detected between transgenic Bt cotton SGK321 and XP188. Hence, Bt protein is not the only incentive resulting in the difference in fungal community composition and diversity, the decrease in biomass between transgenic cotton and untransgenic cotton, different cotton varieties has an effect on them. PMID:25639113

  8. Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas.

    PubMed

    Shen, B-C; Stewart, C N; Zhang, M-Q; Le, Y-T; Tang, Z-X; Mi, X-C; Wei, W; Ma, K-P

    2006-09-01

    Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

  9. 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

  10. 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. PMID:25875245

  11. 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

  12. N-terminal activation is an essential early step in the mechanism of action of the Bacillus thuringiensis Cry1Ac insecticidal toxin.

    PubMed

    Bravo, Alejandra; Sanchez, Jorge; Kouskoura, Thaleia; Crickmore, Neil

    2002-07-01

    A variant form of the Bacillus thuringiensis Cry1Ac toxin that is not cleaved at the N terminus during proteolytic activation with trypsin was found to be incapable of forming pores in Manduca sexta brush border membrane vesicles in vitro and had reduced insecticidal activity in vivo. Binding studies indicated an altered binding pattern of the mutant toxin in that bound toxin could not be fully displaced by a high molar excess of fully trypsin-activated toxin. These results suggest that proteolytic removal of the N-terminal peptide of Cry1Ac is an important step in toxin activation.

  13. Comparison of nutritional quality between Chinese indica rice with sck and cry1Ac genes and its nontransgenic counterpart.

    PubMed

    Li, X; Huang, K; He, X; Zhu, B; Liang, Z; Li, H; Luo, Y

    2007-08-01

    Nutritional assessment of transgenic crops used for human food and animal feed is an important aspect of safety evaluations. An insect-resistant rice (IRR) was generated by the stable insertion of sck, a modified cowpea trypsin inhibitor gene, and cry1Ac, encoding a crystal protein from Bacillus thuringiensis into the genome of a common variety of Chinese indica rice. The composition of the brown and milled rice grain from the resulted IRR line designated Liangyou Kefeng No. 6 was compared with that of the parental rice cultivar Liangyou 2186. Nutrients, including the proximates, amino acids, fatty acids, minerals, and vitamins, were measured. The antinutritive components such as phytic acid, lectin, and trypsin inhibitors were also examined. The data demonstrated that the nutritional quality of both the brown and milled rice grains from the transgenic line was substantially equivalent to that of the nontransgenic counterpart, and measured amounts of nutritional components fell within the range of values reported for other commercial lines.

  14. 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

  15. Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system.

    PubMed

    Wang, Jing; Zhang, Haonan; Wang, Huidong; Zhao, Shan; Zuo, Yayun; Yang, Yihua; Wu, Yidong

    2016-09-01

    Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera.

  16. 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. PMID:25497237

  17. New insight to structure-function relationship of GalNAc mediated primary interaction between insecticidal Cry1Ac toxin and HaALP receptor of Helicoverpa armigera.

    PubMed

    Sengupta, Anindita; Sarkar, Anindya; Priya, Prerna; Ghosh Dastidar, Shubhra; Das, Sampa

    2013-01-01

    Over the last few decades Cry1Ac toxin has been widely used in controlling the insect attack due to its high specificity towards target insects. The pore-forming toxin undergoes a complex mechanism in the insect midgut involving sequential interaction with specific glycosylated receptors in which terminal GalNAc molecule plays a vital role. Recent studies on Cry toxins interactions with specific receptors revealed the importance of several amino acid residues in domain III of Cry1Ac, namely Q509, N510, R511, Y513 and W545, serve as potential binding sites that surround the putative GalNAc binding pocket and mediate the toxin-receptor interaction. In the present study, alanine substitution mutations were generated in the Cry1Ac domain III region and functional significance of those key residues was monitored by insect bioassay on Helicoverpa armigera larvae. In addition, ligand blot analysis and SPR binding assay was performed to monitor the binding characteristics of Cry1Ac wild type and mutant toxins towards HaALP receptor isolated from Helicoverpa armigera. Mutagenesis data revealed that, alanine substitutions in R511, Y513 and W545 substantially impacted the relative affinity towards HaALP receptor and toxicity toward target insect. Furthermore, in silico study of GalNAc-mediated interaction also confirmed the important roles of these residues. This structural analysis will provide a detail insight for evaluating and engineering new generation Cry toxins to address the problem of change in insect behavioral patterns.

  18. 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. PMID:25754522

  19. Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system.

    PubMed

    Wang, Jing; Zhang, Haonan; Wang, Huidong; Zhao, Shan; Zuo, Yayun; Yang, Yihua; Wu, Yidong

    2016-09-01

    Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera. PMID:27343383

  20. Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac).

    PubMed

    Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue; Roelofs, Dick; Chen, Fajun; Zhu-Salzman, Keyan; Liang, Yuyong; Sun, Yucheng; Ge, Feng

    2014-01-01

    The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. High Susceptibility to Cry1Ac and Low Resistance Allele Frequency Reduce the Risk of Resistance of Helicoverpa armigers 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

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

    PubMed

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

    2010-11-01

    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

  7. 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. PMID:26914608

  8. 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. PMID:27532632

  9. Characterization of directly transformed weedy Brassica rapa and introgressed B. rapa with Bt cry1Ac and gfp genes.

    PubMed

    Moon, Hong S; Halfhill, Matthew D; Good, Laura L; Raymer, Paul L; Neal Stewart, C

    2007-07-01

    Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa x B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.

  10. Characterization of directly transformed weedy Brassica rapa and introgressed B. rapa with Bt cry1Ac and gfp genes.

    PubMed

    Moon, Hong S; Halfhill, Matthew D; Good, Laura L; Raymer, Paul L; Neal Stewart, C

    2007-07-01

    Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa x B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds. PMID:17333014

  11. 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.

  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

    ... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into... Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens is exempt from the... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of...

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

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... From Tolerances § 180.1107 Delta endotoxin of Bacillus thuringiensis variety kurstaki encapsulated into... Bacillus thuringiensis variety kurstaki encapsulated into killed Pseudomonas fluorescens is exempt from the... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of...

  14. 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. PMID:26689874

  15. 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

  16. Downregulation and mutation of a Cadherin gene associated with Cry1Ac resistance in the Asian Corn Borer, Ostrinia furnacalis (Guenée).

    PubMed

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

    2014-09-01

    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

  17. 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

  18. Determining the involvement of two aminopeptidase Ns in the resistance of Plutella xylostella to the Bt toxin Cry1Ac: cloning and study of in vitro function.

    PubMed

    Chang, Xiaoli; Wu, Qingjun; Wang, Shaoli; Wang, Ran; Yang, Zhongxia; Chen, Defeng; Jiao, Xiaoguo; Mao, Zhenchuan; Zhang, Youjun

    2012-02-01

    The cloning, expression in vitro, and characterization of two aminopeptidase Ns (APN5s and APN2s) isolated from the midgut of Cry1Ac-resistant (R) and susceptible (S) strains of Plutella xylostella larvae are presented in this paper. The deduced amino acid sequences of APN5s included C-terminal GPI-modification sites, the gluzincin aminopeptidase motif GATEN, and three N-glycosylated sites; those of APN2s had no GPI-modification sites, had gluzincin aminopeptidase motif GAMEN, and had four N-glycosylated sites. O-glycosylated sites were not predicted for either APN. Because APN2R and APN2S cDNAs contained the same nucleotides, only full-length cDNAs encoding APN5R and APN5S were expressed in Trichoplusia ni cells. Far-Western blotting showed that the expressed receptor APN5 bound to the Cry1Ac toxin. An enzyme-specific activity experiment also showed that APN5 genes were expressed in T. ni cells. ELISA revealed no differences in the binding of expression proteins from the resistant and susceptible strain with Cry1Ac. PMID:22371317

  19. 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.

  20. Bacterial community structure in the rhizosphere of a Cry1Ac Bt-brinjal crop and comparison to its non-transgenic counterpart in the tropical soil.

    PubMed

    Singh, Amit Kishore; Rai, Govind Kumar; Singh, Major; Dubey, Suresh Kumar

    2013-11-01

    To elucidate whether the transgenic crop alters the rhizospheric bacterial community structure, a 2-year study was performed with Cry1Ac gene-inserted brinjal crop (Bt) and their near isogenic non-transformed trait (non-Bt). The event of Bt crop (VRBT-8) was screened using an insect bioassay and enzyme-linked immunosorbent assay. Soil moisture, NH4 (+)-N, NO3 (-)-N, and PO4 (-)-P level had non-significant variation. Quantitative polymerase chain reaction revealed that abundance of bacterial 16S rRNA gene copies were lower in soils associated with Bt brinjal. Microbial biomass carbon (MBC) showed slight reduction in Bt brinjal soils. Higher MBC values in the non-Bt crop soil may be attributed to increased root activity and availability of readily metabolizable carbon compounds. The restriction fragment length polymorphism of PCR-amplified rRNA gene fragments detected 13 different bacterial groups with the exclusive presence of β-Proteobacteria, Chloroflexus, Planctomycetes, and Fusobacteria in non-Bt, and Cyanobacteria and Bacteroidetes in Bt soils, respectively, reflecting minor changes in the community structure. Despite the detection of Cry1Ac protein in the rhizospheric soil, the overall impact of Cry1Ac expressing Bt brinjal was less compared to that due to seasonal changes.

  1. 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. PMID:12633848

  2. 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.

  3. 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

  4. Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from Helicoverpa armigera by an improved pull-down assay.

    PubMed

    Zhou, Zishan; Wang, Zeyu; Liu, Yuxiao; Liang, Gemei; Shu, Changlong; Song, Fuping; Zhou, Xueping; Bravo, Alejandra; Soberón, Mario; Zhang, Jie

    2016-08-01

    Cry1Ac toxin-binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull-down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to the LC-MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase-N, alkaline phosphatase, cadherin-like protein, ATP-binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin-binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera. PMID:27037552

  5. Cry1Ac protoxin from Bacillus thuringiensis promotes macrophage activation by upregulating CD80 and CD86 and by inducing IL-6, MCP-1 and TNF-α cytokines.

    PubMed

    Moreno-Fierros, Leticia; García-Hernández, Ana Lilia; Ilhuicatzi-Alvarado, Damaris; Rivera-Santiago, Lucio; Torres-Martínez, Marilú; Rubio-Infante, Néstor; Legorreta-Herrera, Martha

    2013-12-01

    Bacillus thuringiensis Cry1Ac protoxin (pCry1Ac) is a promising mucosal adjuvant, but its action mechanism is unknown. We examined in vivo whether pCry1Ac promotes the activation of macrophages in the peritoneum, spleen and mesenteric lymph nodes or in the lungs and bronchoalveolar lavage after intraperitoneal or intranasal pCry1Ac administration, respectively, in BALB/c mice. pCry1Ac upregulated the costimulatory molecules CD80 and CD86 in these macrophages, but with distinct kinetics. In vitro stimulation of resident macrophages with pCry1Ac upregulated CD80 and CD86 and enhanced the production of the pro-inflammatory cytokines TNF-α, IL-6 and MCP-1. To investigate whether the pCry1Ac-induced activation was mediated through MAPK pathways, we pretreated RAW 264.7 cells with signaling inhibitors of MEK, JNK and p38 MAPKs (PD98059, SP600125 and SB203580, respectively). pCry1Ac-induced upregulation of CD86 and CD80 was partially inhibited by the MEK inhibitor. While LPS-induced upregulation mechanisms of CD80 and CD86 appear to be different; as these were particularly inhibited by MEK and JNK inhibitors, respectively. pCry1Ac-induced IL-6 and MCP-1 production was especially inhibited with the p38 MAPK inhibitor, whereas TNF-α was only slightly inhibited upon treatment with JNK and p38 MAPK inhibitors. Therefore macrophage stimulation with pCry1Ac induced the upregulation of CD80 and CD86, and the production of IL-6, TNF-α and MCP-1, possibly, through the MEK and p38 MAPK pathways. It also promoted the nuclear translocation of NF-κB p50 and p65, the upregulation of MHC-II, and the activation of T CD4+ cells. These results suggest that pCry1Ac induced macrophage activation through mechanisms which differ partially from the LPS-induced. PMID:24157331

  6. Expression of Bacillus thuringiensis delta-endotoxin genes during vegetative growth.

    PubMed Central

    Mettus, A M; Macaluso, A

    1990-01-01

    Bacillus thuringiensis delta-endotoxin (crystal protein) genes are normally expressed only during sporulation. It is possible to produce crystal protein during vegetative growth by placing B. thuringiensis crystal protein genes downstream of a strong vegetative promoter. By removing a possible transcriptional terminator of the tetracycline resistance gene of pBC16 and inserting a multiple cloning site, delta-endotoxin genes can be cloned downstream from the tetracycline resistance gene promoter. This construct allows for readthrough transcription from the strong vegetative promoter. Crystal protein is then produced during vegetative growth as well as during sporulation in both B. thuringiensis and Bacillus megaterium. This construct also allows for production of delta-endotoxin in B. thuringiensis strains that do not normally produce delta-endotoxin because of a defect in sporulation. Images PMID:2160219

  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. PMID:27391960

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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...

  13. 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...

  14. 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...

  15. 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. PMID:27055120

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

    2016-01-01

    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

  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. 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

    ... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated... of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens is... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Delta endotoxin of...

  4. 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

    ... From Tolerances § 180.1108 Delta endotoxin of Bacillus thuringiensis variety San Diego encapsulated... of Bacillus thuringiensis variety San Diego encapsulated into killed Pseudomonas fluorescens is... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Delta endotoxin of...

  5. Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of cry1AC-carrying Brassica rapa subsp. pekinensis.

    PubMed

    Jung, Sera; Park, Semi; Kim, Daeha; Kim, Seung Bum

    2008-02-01

    The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.

  6. Prays oleae midgut putative receptor of Bacillus thuringiensis vegetative insecticidal protein Vip3LB differs from that of Cry1Ac toxin.

    PubMed

    Abdelkefi-Mesrati, Lobna; Rouis, Souad; Sellami, Sameh; Jaoua, Samir

    2009-09-01

    Vegetative insecticidal protein (Vip) is a class of insecticidal proteins produced by many Bacillus thuringiensis strains during their vegetative growth stage. The vip3LB gene of B. thuringiensis strain BUPM95, which encodes a protein active against the Lepidoptera olive tree pathogenic insect Prays oleae, was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed Vip3LB protein, found in the E. coli cytoplasmic fraction, was purified and used to produce anti-Vip3LB antibodies. Using the midgut extract of P. oleae, the purified Vip3LB bound to a 65-kDa protein, whereas Cry1Ac toxin bound to a 210-kDa midgut putative receptor. This result justifies the importance of the biological pest control agent Vip3LB that could be used as another alternative particularly in case of resistance to Cry toxins. PMID:19434523

  7. 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

  8. 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. PMID:19353306

  9. 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

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

    2015-12-01

    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

  10. 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.

  11. 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. PMID:27322533

  12. 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

  13. 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.

  14. Quantitative analysis of fitness costs associated with the development of resistance to the Bt toxin Cry1Ac in Helicoverpa armigera.

    PubMed

    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

  15. Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes.

    PubMed

    Halfhill, Matthew D; Sutherland, Jamie P; Moon, Hong Seok; Poppy, Guy M; Warwick, Suzanne I; Weissinger, Arthur K; Rufty, Thomas W; Raymer, Paul L; Stewart, C Neal

    2005-09-01

    Concerns exist that transgenic crop x weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa x transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations.

  16. 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. PMID:25195461

  17. Differential Role of Manduca sexta Aminopeptidase-N and Alkaline Phosphatase in the Mode of Action of Cry1Aa, Cry1Ab, and Cry1Ac Toxins from Bacillus thuringiensis

    PubMed Central

    Flores-Escobar, Biviana; Rodríguez-Magadan, Hector; Bravo, Alejandra; Soberón, Mario

    2013-01-01

    Aminopeptidase-N (APN1) and alkaline phosphatase (ALP) proteins located in the midgut epithelium of Manduca sexta have been implicated as receptors for Cry1Aa, Cry1Ab, and Cry1Ac insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki. In this study, we analyzed the roles of ALP and APN1 in the toxicity of these three Cry1A proteins. Ligand blot analysis using brush border membrane vesicles of M. sexta showed that Cry1Aa and Cry1Ab bind preferentially to ALP during early instars while binding to APN was observed after the third instar of larval development. Cry1Ac binds to APN throughout all larval development, with no apparent binding to ALP. ALP was cloned from M. sexta midgut RNA and expressed in Escherichia coli. Surface plasmon resonance binding analysis showed that recombinant ALP binds to Cry1Ac with 16-fold lower affinity than to Cry1Aa or Cry1Ab. Downregulation of APN1 and ALP expression by RNA interference (RNAi) using specific double-stranded RNA correlated with a reduction of transcript and protein levels. Toxicity analysis of the three Cry1A proteins in ALP- or APN1-silenced larvae showed that Cry1Aa relies similarly on both receptor molecules for toxicity. In contrast, RNAi experiments showed that ALP is more important than APN for Cry1Ab toxicity, while Cry1Ac relied principally on APN1. These results indicated that ALP and APN1 have a differential role in the mode of action of Cry1A toxins, suggesting that B. thuringiensis subsp. kurstaki produces different Cry1A toxins that in conjunction target diverse midgut proteins to exert their insecticidal effect. PMID:23686267

  18. Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var. tenebrionis.

    PubMed Central

    Carroll, J; Li, J; Ellar, D J

    1989-01-01

    Insecticidal protein delta-endotoxin crystals harvested from sporulated cultures of Bacillus thuringiensis var. tenebrionis contain a major polypeptide of 67 kDa and minor polypeptides of 73, 72, 55 and 46 kDa. During sporulation, only the 73 kDa polypeptide could be detected at stage I. The 67 kDa polypeptide was first detected at stage II and increased in concentration throughout the later stages of sporulation and after crystal release, with a concomitant decrease in the 73 kDa polypeptide. This change could be blocked by the addition of proteinase inhibitors. Trypsin or insect-gut-extract treatment of the delta-endotoxin crystals after solubilization resulted in a cleavage product of 55 kDa with asparagine-159 of the deduced amino acid sequence of the toxin [Höfte, Seurinck, van Houtven & Vaeck (1987) Nucleic Acids Res. 15, 71-83; Sekar, Thompson, Maroney, Bookland & Adang (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7036-7040; McPherson, Perlak, Fuchs, Marrone, Lavrik & Fischhoff (1988) Biotechnology 6, 61-66] at the N-terminus. This polypeptide was found to be as toxic in vivo as native delta-endotoxin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2549968

  19. 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

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

    1996-11-01

    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.

  20. Growth, productivity, and competitiveness of introgressed weedy Brassica rapa hybrids selected for the presence of Bt cry1Ac and gfp transgenes.

    PubMed

    Halfhill, Matthew D; Sutherland, Jamie P; Moon, Hong Seok; Poppy, Guy M; Warwick, Suzanne I; Weissinger, Arthur K; Rufty, Thomas W; Raymer, Paul L; Stewart, C Neal

    2005-09-01

    Concerns exist that transgenic crop x weed hybrid populations will be more vigorous and competitive with crops compared with the parental weed species. Hydroponic, glasshouse, and field experiments were performed to evaluate the effects of introgression of Bacillus thuringiensis (Bt) cry1Ac and green fluorescent protein (GFP) transgenes on hybrid productivity and competitiveness in four experimental Brassica rapa x transgenic Brassica napus hybrid generations (F1, BC1F1, BC2F1 and BC2F2). The average vegetative growth and nitrogen (N) use efficiency of transgenic hybrid generations grown under high N hydroponic conditions were lower than that of the weed parent (Brassica rapa, AA, 2n = 20), but similar to the transgenic crop parent, oilseed rape (Brassica napus, AACC, 2n = 38). No generational differences were detected under low N conditions. In two noncompetitive glasshouse experiments, both transgenic and nontransgenic BC2F2 hybrids had on average less vegetative growth and seed production than B. rapa. In two high intraspecific competition field experiments with varied herbivore pressure, BC2F2 hybrids produced less vegetative dry weight than B. rapa. The competitive ability of transgenic and nontransgenic BC2F2 hybrids against a neighbouring crop species were quantified in competition experiments that assayed wheat (Triticum aestivum) yield reductions under agronomic field conditions. The hybrids were the least competitive with wheat compared with parental Brassica competitors, although differences between transgenic and nontransgenic hybrids varied with location. Hybridization, with or without transgene introgression, resulted in less productive and competitive populations. PMID:16101783

  1. Efficacy of Soybean's Event DAS-81419-2 Expressing Cry1F and Cry1Ac to Manage Key Tropical Lepidopteran Pests Under Field Conditions in Brazil.

    PubMed

    Marques, L H; Castro, B A; Rossetto, J; Silva, O A B N; Moscardini, V F; Zobiole, L H S; Santos, A C; Valverde-Garcia, P; Babcock, J M; Rule, D M; Fernandes, O A

    2016-08-01

    Bacillus thuringiensis (Bt) event DAS-81419-2 (Conkesta technology) in soybean, Glycine max (L.) Merrill, expresses Cry1F and Cry1Ac proteins to provide protection from feeding by several lepidopteran pests. A total of 27 field experiments across nine locations were conducted from 2011 to 2015 in southern and central Brazil to characterize the efficacy of DAS-81419-2 soybean infested with Anticarsia gemmatalis Hübner (Lepidoptera: Erebidae), Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae), Heliothis virescens (F.) (Lepidoptera: Noctuidae), and Spodoptera cosmioides (Walker) (Lepidoptera: Noctuidae) during vegetative (V4) and reproductive (R2 and R4) crop developmental stages. The efficacy of DAS-81419-2 was compared to that of a non-Bt isogenic variety managed with or without applications of commercial foliar insecticides for lepidopteran control. DAS-81419-2 soybean consistently experienced defoliation levels of 0.5% or less (compared with 20.05-56.74% in the non-Bt, nonsprayed treatment) and larval survival of < 0.1% in all four species across the vegetative and reproductive plant stages evaluated. The efficacy of DAS-81419-2 was significantly higher than commercial foliar insecticides applied to the non-Bt variety. DAS-81419-2 soybeans containing two highly effective Bt proteins are expected to be a more robust IRM tool compared to single-trait Bt technologies. The consistent efficacy of DAS-81419-2 soybeans across years, locations, and crop stages suggests that it will be a valuable product for management of hard-to-control key lepidopteran pests in South American soybean production. PMID:27401112

  2. 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. PMID:26928903

  3. The interaction of two-spotted spider mites, Tetranychus urticae Koch, with Cry protein production and predation by Amblyseius andersoni (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize.

    PubMed

    Guo, Yan-Yan; Tian, Jun-Ce; Shi, Wang-Peng; Dong, Xue-Hui; Romeis, Jörg; Naranjo, Steven E; Hellmich, Richard L; Shelton, Anthony M

    2016-02-01

    Crops producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), are an important tool for managing lepidopteran pests on cotton and maize. However, the effects of these Bt crops on non-target organisms, especially natural enemies that provide biological control services, are required to be addressed in an environmental risk assessment. Amblyseius andersoni (Acari: Phytoseiidae) is a cosmopolitan predator of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), a significant pest of cotton and maize. Tri-trophic studies were conducted to assess the potential effects of Cry1Ac/Cry2Ab cotton and Cry1F maize on life history parameters (survival rate, development time, fecundity and egg hatching rate) of A. andersoni. We confirmed that these Bt crops have no effects on the biology of T. urticae and, in turn, that there were no differences in any of the life history parameters of A. andersoni when it fed on T. urticae feeding on Cry1Ac/Cry2Ab or non-Bt cotton and Cry1F or non-Bt maize. Use of a susceptible insect assay demonstrated that T. urticae contained biologically active Cry proteins. Cry proteins concentrations declined greatly as they moved from plants to herbivores to predators and protein concentration did not appear to be related to mite density. Free-choice experiments revealed that A. andersoni had no preference for Cry1Ac/Cry2Ab cotton or Cry1F maize-reared T. urticae compared with those reared on non-Bt cotton or maize. Collectively these results provide strong evidence that these crops can complement other integrated pest management tactics including biological control. PMID:26545599

  4. The interaction of two-spotted spider mites, Tetranychus urticae Koch, with Cry protein production and predation by Amblyseius andersoni (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize.

    PubMed

    Guo, Yan-Yan; Tian, Jun-Ce; Shi, Wang-Peng; Dong, Xue-Hui; Romeis, Jörg; Naranjo, Steven E; Hellmich, Richard L; Shelton, Anthony M

    2016-02-01

    Crops producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), are an important tool for managing lepidopteran pests on cotton and maize. However, the effects of these Bt crops on non-target organisms, especially natural enemies that provide biological control services, are required to be addressed in an environmental risk assessment. Amblyseius andersoni (Acari: Phytoseiidae) is a cosmopolitan predator of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), a significant pest of cotton and maize. Tri-trophic studies were conducted to assess the potential effects of Cry1Ac/Cry2Ab cotton and Cry1F maize on life history parameters (survival rate, development time, fecundity and egg hatching rate) of A. andersoni. We confirmed that these Bt crops have no effects on the biology of T. urticae and, in turn, that there were no differences in any of the life history parameters of A. andersoni when it fed on T. urticae feeding on Cry1Ac/Cry2Ab or non-Bt cotton and Cry1F or non-Bt maize. Use of a susceptible insect assay demonstrated that T. urticae contained biologically active Cry proteins. Cry proteins concentrations declined greatly as they moved from plants to herbivores to predators and protein concentration did not appear to be related to mite density. Free-choice experiments revealed that A. andersoni had no preference for Cry1Ac/Cry2Ab cotton or Cry1F maize-reared T. urticae compared with those reared on non-Bt cotton or maize. Collectively these results provide strong evidence that these crops can complement other integrated pest management tactics including biological control.

  5. 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

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false CryIA(c) and CryIC derived delta... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  6. 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

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false CryIA(c) and CryIC derived delta... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  7. 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

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false CryIA(c) and CryIC derived delta... RESIDUES IN FOOD Exemptions From Tolerances § 180.1154 CryIA(c) and CryIC derived delta-endotoxins of... plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  8. Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc-Gamma-II/and Fc-Gamma-III Receptors Associated with IgG in the Intestinal Epithelium of Mice.

    PubMed

    Moreno-Fierros, L; Verdín-Terán, S L; García-Hernández, A L

    2015-07-01

    In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ-R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ-RII (CD32) and Fcγ-RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co-localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross-immunoprecipitation results with anti-CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ-RII and Fcγ-RIII and contained IgG associated with Fcγ-RII/RIII. Using anti-CD32 and anti-CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ-RII and Fcγ-RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti-CD16/32, anti-CD16 and anti-CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ-RII and Fcγ-RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice. PMID:25904149

  9. 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

    ...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the expression plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  10. 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

    ...-endotoxins of Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the... Bacillus thuringiensis var. kurstaki encapsulated in killed Pseudomonas fluorescens, and the expression plasmid and cloning vector genetic constructs. CryIA(c) and CryIC derived delta-endotoxins of...

  11. 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

  12. 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.

  13. 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. PMID

  14. 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.

  15. 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.

  16. 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. PMID:25899034

  17. 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.

  18. 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

  19. DELTAE

    SciTech Connect

    Ward, W.C. )

    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.

  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. PMID:25720597

  1. 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.

  2. Extracorporeal adsorption of endotoxin.

    PubMed

    Staubach, K H; Rosenfeldt, J A; Veit, O; Bruch, H P

    1997-02-01

    In a porcine endotoxin shock model using a continuous intravenous endotoxin infusion of 250 ng/kg body weight per hour, the cardiorespiratory and hematologic parameters were studied while applying a new on-line polymyxin B immobilized adsorption system. This preliminary report shows that the new adsorbent can remove endotoxin selectively from the circulation and confers a good amount of protection from endotoxin-induced cardiopulmonary decompensation as well as hematologic alterations. Survival time could be extended from 216 min to 313 min. Whereas cardiac output and mean arterial pressure declined critically after 3 h in the controls, the treated group remained stable for another 3 h. These data show that endotoxin adsorption by polymyxin B coupled covalently to acrylic spheres as an adjunctive on-line measure in the septic syndrome seems feasible. PMID:10225785

  3. Endotoxin in meningococcal infections.

    PubMed Central

    Tubbs, H R

    1980-01-01

    26 children with meningococcal infections were studied to find out the relationaship between plasma and cerebrospinal fluid levels of endotoxin, the clinical outcome, the level of antigen in plasma and cerebrospinal fluid, and indices of complement activation and disseminated intravascular coagulation. No association was found between endotoxin levels and the other factors. A high cerebrospinal fluid antigen level in patients with meningitis was associated with a poor prognosis. PMID:6776899

  4. 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.

  5. Antisera-mediated in vivo reduction of Cry 1Ac toxicity against Helicoverpa armigera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A functional assessment of Bacillus thuringiensis (Bt) toxin receptors in the midgut of lepidopteran insects will facilitate understanding of the toxin mode of action and provide effective strategies to counter the development of resistance. In this study, we produced anti-aminopeptidase (APN) and ...

  6. 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.

  7. Perioperative anti-endotoxin strategies.

    PubMed

    Houdijk, A P; Meijer, C; Cuesta, M A; Meyer, S; Van Leeuwen, P A

    1997-01-01

    Lipopolysaccharides from the outer membrane of Gram-negative bacteria are potent stimuli for the production of numerous cytokines by the immune cells. The systemic inflammatory response to these gut-derived endotoxins is therefore dependent on the responsiveness of the immune system. This paper presents results on anti-endotoxin strategies and the responsiveness to endotoxin in animal models of liver failure. Following partial hepatectomy in the normal rat, anti-endotoxin treatment using the enteral endotoxin binder cholestyramine and the bactericidal permeability-increasing protein showed beneficial effects in terms of reducing the exaggerated metabolic and inflammatory responses. Similar beneficial effects of gut endotoxin restriction were found in bile duct ligated rats subjected to a laparotomy. The beneficial effects of anti-endotoxin strategies in these models were explained by completely different mechanisms. In partial hepatectomized rats the effects were explained by the direct inhibition of the stimulatory action of endotoxin on immune cells preventing an exaggerated inflammatory response. In contrast, in postoperative BDL rats the effects of anti-endotoxin therapy were explained by the restoration of endotoxin sensitivity of the immune cells resulting in an inflammatory response necessary for an adequate reaction to surgery. These different mechanism will be discussed in the light of the phenomenon of endotoxin tolerance.

  8. 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. PMID:16172896

  9. Anti-endotoxin vaccines

    PubMed Central

    Cross, Alan S

    2014-01-01

    Gram-negative bacterial (GNB) infections are a leading cause of serious infections both in hospitals and the community. The mortality remains high despite potent antimicrobials and modern supportive care. In the last decade invasive GNB have become increasingly resistant to commonly used antibiotics, and attempts to intervene with novel biological therapies have been unsuccessful. Earlier studies with antibodies directed against a highly conserved core region in the GNB lipopolysaccharide (LPS, or endotoxin) suggested that this approach may have therapeutic benefit, and led to the development of a subunit vaccine that has progressed to phase 1 clinical testing. Since only a few serogroups of GNB cause bacteremia, O-specific vaccines had been developed, but these were not deployed because of the availability of other therapeutic options at the time. Given the likelihood that new antibiotics will not be soon available, the development of vaccines and antibodies directed against endotoxin, both O and core antigens, deserves a “second look”. PMID:23974910

  10. 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

  11. Endotoxin hitchhiking on polymer nanoparticles.

    PubMed

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

    2016-07-15

    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 × 10(5) 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

  12. Endotoxin hitchhiking on polymer nanoparticles.

    PubMed

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

    2016-07-15

    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 × 10(5) 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

  13. Chylomicrons enhance endotoxin excretion in bile.

    PubMed Central

    Read, T E; Harris, H W; Grunfeld, C; Feingold, K R; Calhoun, M C; Kane, J P; Rapp, J H

    1993-01-01

    Chylomicrons prevent endotoxin toxicity and increase endotoxin uptake by hepatocytes. As a consequence, less endotoxin is available to activate macrophages, thereby reducing tumor necrosis factor secretion. To determine whether the chylomicron-mediated increase in hepatocellular uptake of endotoxin results in increased endotoxin excretion into bile, we examined bile after endotoxin administration. A sublethal dose (7 micrograms/kg) of 125I-endotoxin was incubated with either rat mesenteric lymph containing nascent chylomicrons (500 mg of chylomicron triglyceride per kg of body weight) or an equal volume of normal saline (controls) for 3 h and then infused into male Sprague-Dawley rats. Bile samples were collected via a common bile duct catheter for 24 h. Infusion of endotoxin incubated with chylomicrons increased biliary excretion of endotoxin by 67% at 3 h (P < or = 0.006) and by 20% at 24 h (P < or = 0.01) compared with infusion of endotoxin incubated in saline. Endotoxin activity, as measured by the Limulus assay, was not detected in the bile of test animals. However, endotoxin activity was detected after hot phenol-water extraction of bile, demonstrating that endotoxin is inactive in the presence of bile but retains bioactivity after hepatic processing. Since the majority of an intravenous endotoxin load has been shown to be cleared by the liver, acceleration of hepatocyte clearance and biliary excretion of endotoxin may represent a component of the mechanism by which chylomicrons protect against endotoxin-induced lethality. PMID:8335381

  14. Endotoxin Deactivation by Transient Acidification

    PubMed Central

    Ribeiro, Melina M.; Xu, Xiumin; Klein, Dagmar; Kenyon, Norma S; Ricordi, Camillo; Felipe, Maria Sueli S.; Pastori, Ricardo L.

    2015-01-01

    Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide-endotoxin found in the outer bacterial membrane. Endotoxin could interfere with in vitro biological assays and is the major pathological factor, which must be removed or inactivated before in vivo administration. Here we describe a one-step protocol in which the endotoxin activity on recombinant proteins is remarkably reduced by transient exposure to acidic conditions. Maximum endotoxin deactivation occurs at acidic pH below their respective isoelectric point (pI). This method does not require additional protein purification or separation of the protein from the endotoxin fraction. The endotoxin level was measured both in vitro and in vivo. For in vitro assessment we have utilized Limulus Amebocyte Lysate method for in vivo the pyrogenic test. We have tested the above-mentioned method with 5 different recombinant proteins including a monoclonal antibody clone 5c8 against CD154 produced by hybridomas. More than 99% of endotoxin was deactivated in all of the proteins, the recovery of the protein after deactivation varied between maximum 72.9 and minimum 46.8%. The anti CD154 clone 5c8 activity remained unchanged as verified by the measurement of binding capability to activated lymphocytes. Furthermore, the effectiveness of this method was not significantly altered by urea, commonly used in protein purification. This procedure provides a simple and cost-efficient way to reduce the endotoxin activity in antibodies and recombinant proteins. PMID:20412635

  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. 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 ...

  17. 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...

  18. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. 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. PMID:23934306

  1. 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.

  2. Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

    PubMed Central

    Hua, Gang; Masson, Luke; Jurat-Fuentes, Juan Luis; Schwab, George; Adang, Michael J.

    2001-01-01

    Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured 125I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for 125I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit 125I-Cry1Ab binding to BBMV. Cry1F inhibited 125I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins. PMID:11157257

  3. 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....

  4. 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....

  5. 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....

  6. 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....

  7. 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....

  8. Endotoxins and other sepsis triggers.

    PubMed

    Opal, Steven M

    2010-01-01

    Endotoxin, or more accurately termed bacterial lipopolysaccharide (LPS), is recognized as the most potent microbial mediator implicated in the pathogenesis of sepsis and septic shock. Yet despite its discovery well over a century ago, the fundamental role of circulating endotoxin in the blood of most patients with septic shock remains enigmatic and a subject of considerable controversy. LPS is the most prominent 'alarm molecule' sensed by the host's early warning system of innate immunity presaging the threat of invasion of the internal milieu by Gram-negative bacterial pathogens. In small doses within a localized tissue space, LPS signaling is advantageous to the host in orchestrating an appropriate antimicrobial defense and bacterial clearance mechanisms. Conversely, the sudden release of large quantities of LPS into the bloodstream is clearly deleterious to the host, initiating the release of a dysregulated and potentially lethal array of inflammatory mediators and procoagulant factors in the systemic circulation. The massive host response to this single bacterial pattern recognition molecule is sufficient to generate diffuse endothelial injury, tissue hypoperfusion, disseminated intravascular coagulation and refractory shock. Numerous attempts to block endotoxin activity in clinical trials with septic patients have met with inconsistent and largely negative results. Yet the groundbreaking discoveries within the past decade into the precise molecular basis for LPS-mediated cellular activation and tissue injury has rekindled optimism that a new generation of therapies that specifically disrupt LPS signaling might succeed. Other microbial mediators found in Gram-positive bacterial and viral and fungal pathogens are now appreciated to activate many of the same host defense networks induced by LPS. This information is providing novel interventions in the continuing effots to improve the care of septic patients.

  9. 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.

  10. Addressing endotoxin issues in bioengineered heparin.

    PubMed

    Suwan, Jiraporn; Torelli, Amanda; Onishi, Akihiro; Dordick, Jonathan S; Linhardt, Robert J

    2012-01-01

    Heparin is a widely used clinical anticoagulant that is prepared from pig intestine. A contamination of heparin in 2008 has led to a reexamination of animal-derived pharmaceuticals. A bioengineered heparin prepared by bacterial fermentation and chemical and enzymatic processing is currently under development. This study examines the challenges of reducing or removing endotoxins associated with this process that are necessary to proceed with preclinical in vivo evaluation of bioengineered heparin. The current process is assessed for endotoxin levels, and strategies are examined for endotoxin removal from polysaccharides and enzymes involved in this process. PMID:23586950

  11. 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.

  12. 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

  13. 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

    2016-05-01

    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. 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.

  14. 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

    2016-05-01

    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. 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. PMID:27120167

  15. 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 ...

  16. Mississippi Delta

    Atmospheric Science Data Center

    2014-05-15

    article title:  The Mississippi Delta     Left: True Color Image ... Imaging SpectroRadiometer (MISR) images of the Mississippi delta were acquired on April 26, 2000. The true color image displays the ...

  17. 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 ...

  18. Citalopram reduces endotoxin-induced fatigue.

    PubMed

    Hannestad, Jonas; DellaGioia, Nicole; Ortiz, Nyrma; Pittman, Brian; Bhagwagar, Zubin

    2011-02-01

    Increased levels of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-6 (IL-6) may play a role in depression. Mild depressive-like symptoms can be induced in humans through activation of the innate immune system with endotoxin. Whether preventive treatment with antidepressants can reduce endotoxin-induced symptoms has never been tested. In a double-blind, randomized, placebo-controlled, cross-over study, we administered intravenous low-dose endotoxin (0.8 ng/kg) or placebo to 11 healthy subjects who had received oral pre-treatment with citalopram (10 mg twice a day) or placebo for 5 days. The Montgomery-Åsberg Depression Rating Scale, the State and Trait Anxiety Inventory, and a visual analog scale were used to measure depressive and anxiety symptoms and social anhedonia. Serum levels of TNF and IL-6 were measured with immunoassays. Compared to placebo, endotoxin administration increased serum levels of TNF and IL-6, and caused mild depressive-like symptoms, in particular lassitude and social anhedonia. While citalopram pre-treatment had no effect on the innate immune response to endotoxin, it reduced the endotoxin-induced MADRS total score by 50%, with a moderate effect size (Cohen's d=0.5). Most of the MADRS total score was due to the lassitude item, and citalopram pre-treatment specifically reduced endotoxin-induced lassitude with a large effect size (Cohen's d=0.9). These results suggest that subchronic pre-treatment with the serotonin-reuptake inhibitor citalopram blunts mood symptoms induced by acute immune system activation with endotoxin without inhibiting the peripheral immune response.

  19. 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.

  20. 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.

  1. Endotoxin Induced Disseminated Intravascular Coagulation in Cattle

    PubMed Central

    Thomson, G. W.; McSherry, B. J.; Valli, V. E. O.

    1974-01-01

    Endotoxin administered intravenously to a group of four calves resulted in disseminated intravascular coagulation. A sublethal dose of piromen, a commercially available Pseudomonas spp endotoxin, was used. Serial measurements of total plasma fibrinogen, soluble fibrin levels, ethanol gelation tests, protamine sulfate tests, fibrinogen-fibrin-related antigen (FR-antigen) and prothrombin and thrombin times were done. Initial depression of plasma fibrinogen with a nadir of about 40% of pre-endotoxin levels at eight to 11 hours post-endotoxin (+8 to +11 hours) followed by an overcompensation to 180% at +60 to +108 hours was shown. Soluble fibrin was demonstrated in plasma from +2 to +22 hours with a peak of 100-114 mg/100 ml at +4 to +9 hours. Positive plasma ethanol gelation and protamine sulfate tests, as well as the presence of serum FR-antigen, occurred consistently following endotoxin administration. Significant increases in prothrombin times (PT) from +4 to +40 hours and in thrombin times (TT) from +4 to +16 hours were demonstrated. The peak increase of PT at +8 to +10 hours was 180%. The peak increase of TT at +6 to +9 hours was 260-290%. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:4279765

  2. Studies with Radioactive Endotoxin II. Clearance of 3H-Labelled Endotoxin from the Blood of Calves

    PubMed Central

    Maxie, M. G.; Valli, V. E. O.; Lumsden, J. H.

    1974-01-01

    The clearance of 3H-labelled Pseudomonas endotoxin from the blood was studied in a nontolerant and in an endotoxin tolerant state. Calves were rendered tolerant to the toxic effects of the endotoxin by four daily intravenous injections of endotoxin at the dose rate of 5 µg/kg body weight. Clearance of 3H-endotoxin from the blood of nontolerant calves occurred more slowly than did clearance of 51Cr-endotoxin and was not significantly (P<0.05) affected by the development of tolerance. The lungs and liver were the major organs involved in the clearance of 3H-endotoxin from the blood of calves. Leukocytes and erythrocytes, but not platelets, were shown to participate in endotoxin clearance in calves. 3H2O, the control substance used in calves, was not concentrated within any particular organ but rapidly equilibrated with total body water and was slowly excreted. PMID:4279755

  3. 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.

  4. Longitudinal study of dust and airborne endotoxin in the home.

    PubMed

    Park, J H; Spiegelman, D L; Burge, H A; Gold, D R; Chew, G L; Milton, D K

    2000-11-01

    To characterize the seasonal variability of endotoxin levels, we measured endotoxin in dust from the bed, bedroom floor, and kitchen floor in 20 homes, and in air from the bedroom in 15 of the homes. All homes were located in the greater Boston, Massachusetts, area and were sampled each month from April 1995 to June 1996. Outdoor air was collected at two locations. We found greater within-home than between-home variance for bedroom floor, kitchen floor, and airborne endotoxin. However, the reverse was true for bed dust endotoxin. Thus, studies using single measurements of dust endotoxin are most likely to reliably distinguish between homes if bed dust is sampled. Dust endotoxin levels were not significantly associated with airborne endotoxin. Airborne endotoxin was significantly (p = 0. 04) and positively associated with absolute humidity in a mixed-effect model adjusting for a random home effect and fixed effect of sampling month and home characteristics. This finding implies that indoor humidity may be an important factor controlling endotoxin exposure. We found a significant (p < 0.05) seasonal effect in kitchen floor dust (spring > fall) and bedroom airborne endotoxin (spring > winter), but not in the other indoor samples. We found significant seasonal pattern in outdoor airborne endotoxin (summer > winter). PMID:11102291

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

    PubMed

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

    2013-10-01

    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.

  6. 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.

  7. 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. PMID:23908000

  8. [The role of bacterial endotoxins, receptors and cytokines in the pathogenesis of septic (endotoxin) shock].

    PubMed

    Lazarov, S; Balutsov, M; Ianev, E

    2000-01-01

    Sepsis, resistant to therapy, results in the development of septic (endotoxin) shock. The latter is caused by the endotoxins of different Gram-negative bacteria. Endotoxin (bacterial lipopdisacharide--LPS) interacts with cells through specific membrane or plasma soluble endotoxin receptors (sCD14, mlD14, LBP, CD13/CD14, CD16, CD116/CD18, L-selectin, etc.). Endotoxin interaction with the mCD14 receptor of the monocytes, macrophages and the neutrophils results in the production of a number of proinflammatory cytokines--tumor necrosis factor alpha (TNF alpha), interleukines 1 and 6 (IL-1 and IL-6, etc), antiinflammatory cytokines--interleukines 10 and 12 (IL-10 and IL-12), cell adhesion molecules (P-selectin, E-selectin, ICAM-1, VCAM-1, etc.) and inducible enzymes: inducible NO synthase (iNOS), inducible phospholipase A2 (cPL-A2), inducible cyclooxygenase (COX-2). All pathologic processes in the structure and function of human body during endotoxin shock are a result of the disbalance of a number of mediators with a proinflammatory and antiinflammatory effects.

  9. 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

  10. Removal of endotoxin from dairy wastewater

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Bronchial Venular Leakage During Endotoxin Shock

    PubMed Central

    Pietra, G. G.; Szidon, J. P.; Carpenter, H. A.; Fishman, A. P.

    1974-01-01

    The pulmonary effects of endotoxin shock were investigated in dogs by a combination of anatomic and physiologic technics. Shock was produced in 14 dogs by injecting Escherichia coli lipopolysaccharide intravenously. Three dogs in hypovolemic shock and 6 untreated dogs served as controls. Colloidal carbon was injected intravenously to detect sites of pathologic increase in vascular permeability. During the first hour of endotoxin shock, bronchial venules allowed carbon and blood elements to traverse their walls, whereas no leakage of these large particles or ultrastructural changes could be detected in the alveolar walls. Only after the first hour was bronchial venular leakage accompanied by focal degenerative changes in the alveolar endothelium, focal interstitial edema in the alveolar septum and sequestration of damaged leukocytes in the alveolar capillaries. In contrast to these observations in endotoxin shock, control dogs in hypovolemic shock did not show bronchial venous leakage. Our findings suggest that the leakage of bronchial venules may be involved in the pathogenesis of pulmonary interstitial edema caused by endotoxin shock. ImagesFig 2Fig 3Fig 4Fig 1 PMID:4611225

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Predictors of airborne endotoxin concentrations in inner city homes.

    PubMed

    Mazique, D; Diette, G B; Breysse, P N; Matsui, E C; McCormack, M C; Curtin-Brosnan, J; Williams, D L; Peng, R D; Hansel, N N

    2011-05-01

    Few studies have assessed in home factors which contribute to airborne endotoxin concentrations. In 85 inner city Baltimore homes, we found no significant correlation between settled dust and airborne endotoxin concentrations. Certain household activities and characteristics, including frequency of dusting, air conditioner use and type of flooring, explained 36-42% of the variability of airborne concentrations. Measurements of both airborne and settled dust endotoxin concentrations may be needed to fully characterize domestic exposure in epidemiologic investigations. PMID:21429483

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Metabolic profiling based on LC/MS to evaluate unintended effects of transgenic rice with cry1Ac and sck genes.

    PubMed

    Chang, Yuwei; Zhao, Chunxia; Zhu, Zhen; Wu, Zeming; Zhou, Jia; Zhao, Yanni; Lu, Xin; Xu, Guowang

    2012-03-01

    As a primary characteristic of substantial equivalence, the evaluation of unintended effects of genetically modified plants has been evolving into an important field of research. In this study, a metabolic profiling method for rice seeds was developed using rapid resolution liquid chromatography/quadrupole time-of-flight mass spectrometry. The analytical properties of the method, including the linearity, reproducibility, intra-day precision and inter-day precision, were investigated and were found to be satisfactory. The method was then applied to investigate the differences between transgenic rice and its native counterparts, in addition to the differences found between native rice with different sowing dates or locations. Global metabolic phenotype differences were visualized, and metabolites from different discriminated groups were discovered using multivariate data analysis. The results indicated that environmental factors played a greater role than gene modification for most metabolites, including tryptophan, 9,10,13-trihydroxyoctadec-11-enoic acid, and lysophosphatidylethanolamine 16:0. The concentrations of phytosphingosine, palmitic acid, 5-hydroxy-2-octadenoic acid and three other unidentified metabolites varied slightly due to gene modification. PMID:22271304

  20. Effect of hypertriglyceridemia on endotoxin responsiveness in humans.

    PubMed

    van der Poll, T; Braxton, C C; Coyle, S M; Boermeester, M A; Wang, J C; Jansen, P M; Montegut, W J; Calvano, S E; Hack, C E; Lowry, S F

    1995-09-01

    Triglyceride-rich lipoproteins can inhibit endotoxin activity in vitro and in rodents. We sought to determine whether Intralipid, a triglyceride-rich fat emulsion which in contact with plasma functions similarly to endogenous lipoproteins, can alter the human response to endotoxin. Intralipid inhibited endotoxin-induced cytokine production in human whole blood in vitro in a dose-dependent manner, with maximal inhibition (up to 70%) being achieved at a concentration of 10 g/liter. In healthy men, a bolus intravenous injection of endotoxin (lot EC-5; 20 U/kg of body weight) was given midway through a 4-h infusion (125 ml/h) of either 5% glucose (n = 5) or 20% Intralipid (n = 5). The infusion of Intralipid led to an increase in triglyceride levels in serum from 95 +/- 16 to 818 +/- 135 mg/dl prior to endotoxin administration, i.e., levels that importantly reduced cytokine production in endotoxin-stimulated whole blood. However, in vivo hypertriglyceridemia did not influence inflammatory responses to endotoxin (fever, release of tumor necrosis factor and soluble tumor necrosis factor receptors, and leukocytosis) or even potentiated endotoxin responses (release of interleukins 6 and 8 and neutrophil degranulation). Hypertriglyceridemia does not inhibit the in vivo responses to endotoxin in humans.

  1. 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.

  2. Endotoxin and cancer chemo-prevention.

    PubMed

    Mastrangelo, Giuseppe; Fadda, Emanuela; Cegolon, Luca

    2013-10-01

    Reduced rates of lung cancer have been observed in several occupational groups exposed to high levels of organic dusts contaminated by endotoxin. The underlying anti-neoplastic mechanism of endotoxin may be an increased secretion of endogenous anti-neoplastic mediators and activation of the toll-like receptors (TLR). A detoxified endotoxin derivative, Monophosphoryl Lipid A (MPL(®)) is marketed in Europe since 1999 as part of the adjuvant systems in allergy vaccines for treatment of allergic rhino-conjunctivitis and allergic asthma. Over 200,000 patients have used them to date (nearly 70% in Germany). Since detailed exposure (MPL(®) dose and timing of administration) and individual data are potentially available, an observational follow-up study could be conducted in Germany to investigate the protective effect of MPL(®) against cancer, comparing cancer incidence in two groups of patients with allergic rhinitis: those treated with allergoids plus MPL(®) and those treated with a vaccine including the same allergoids but not MPL(®). The protective effect of MPL(®) could be quantified in ever and never smokers. If this proposed observational study provides evidence of protective effects, MPL(®) could be immediately used as a chemo-preventive agent since it is already in use as adjuvant in human vaccines against cancer.

  3. Exposure to inhalable dust and endotoxins in agricultural industries.

    PubMed

    Spaan, Suzanne; Wouters, Inge M; Oosting, Isabella; Doekes, Gert; Heederik, Dick

    2006-01-01

    Endotoxin is a well-known bacterial toxin that causes several health effects. Animal faeces and plant materials contaminated with bacteria have been identified as important determinants of organic dust related endotoxin exposure. Although high exposure to organic dust and endotoxins has been described regularly in agricultural industries, a detailed overview of levels of airborne exposure to endotoxins in the agricultural industry, as well as a systematic comparison between several specific branches using the same exposure assessment protocols are lacking. In this study, personal endotoxin exposure in a broad spectrum of agricultural industries was investigated and possible determinants of exposure were explored. 601 personal inhalable dust samples were taken in 46 companies of three agricultural industrial sectors: grains, seeds and legumes sector (GSL), horticulture sector (HC) and animal production sector (AP), with 350 participating employees. Dust and endotoxin levels were determined gravimetrically and by using the Limulus Amoebocyte Lysate (LAL) assay, respectively. Basic descriptive analysis and elaborate analysis of variance were performed. Mean exposure levels were high, with large differences between sectors and between companies within the sectors. Highest dust and endotoxin exposures were found in companies of the GSL sector. In all three sectors exposure was higher in the primary production part compared to the (industrial) products processing part of the sector. The Dutch proposed health based occupational exposure limit (50 EU m(-3)) and temporary legal limit (200 EU m(-3)) for endotoxin were often exceeded. Differences in exposure between workers were larger than the day-to-day variability. Identified determinants increasing exposure levels were company, dustiness of the product and contact with animals/faeces. 'Wet' processes resulted in less dusty working environments and thus lowered endotoxin exposure. Overall, exposure to endotoxins over the

  4. 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.

  5. 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, ...

  6. 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.

  7. [Exposure to endotoxins in the environment. Occurrence and health hazards].

    PubMed

    Rix, B A

    1997-04-21

    Endotoxins are lipopolysaccharides from the outer cell wall of Gram-negative bacteria. Exposure to endotoxins can take place in industries where organic material is handled, in agriculture, in garbage handling, and sewage treatment. Byssinosis defined as Monday chest tightness and slight dyspnoea in the work place has been related to endotoxin exposure in cotton mills, but studies indicate that similar symptoms may be found in other work places. Other symptoms are: Headache, nausea, gastrointestinal symptoms and influenza-like symptoms. Several studies have shown a decrease in FEV1 following exposure to endotoxins. The relationship between exposure to organic dust, microorganisms, endotoxins and other chemicals in the work place and disease needs further research.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. [Biochemistry, molecular mechanism of action and biological effects of endotoxin].

    PubMed

    Burgmann, H; Breyer, S

    1995-01-01

    This review is a brief attempt at providing an overview of a subject of enormous complexity-endotoxins and the mediators associated with its biological effects. More specifically it deals with biochemistry and biology of endotoxin, detection of endotoxin with the Limulus amebocyte lysate test, the molecular mechanisms and biological effects, and in the last part with future aspects of therapeutical strategies. It seems certain that the subject will become even more complex and possibly controversial as scientific knowledge further involves. However, because of the high mortality rate of patients suffering from gram-negative sepsis all efforts have to be made to find effective therapeutical strategies.

  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. 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.

  15. Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes.

    PubMed

    Adamczyk, J J; Adams, L C; Hardee, D D

    2001-12-01

    Examination of commercial Cry1Ac transgenic Bacillus thuringiensis Berliner (Bt) cotton varieties (Bollgard, Monsanto, St. Louis, MO) and an experimental Cry1Ac + Cry2Ab transgenic Bt cotton variety (Bollgard II, Monsanto) for lepidopteran field efficacy was conducted during the 2000 growing season. In addition, a commercially available (Envirologix, Portland, ME) quantification assay (ELISA) was used to measure and profile the expression levels of Cry proteins in two of these varieties ['DP50B, Bollgard'; 'DP50BII, Bollgard II' (Delta & Pine Land, Scott, MS)]. Populations of beet army worms, Spodoptera exigua (Hübner), and soybean loopers, Pseudoplusia includens (Walker), were significantly lower (P < 0.05) in Bollgard II plots compared with Bollgard. Population numbers for fall army worms, Spodoptera frugiperda (J. E. Smith), and salt marsh caterpillars, Estigmene acrea (Drury), were lower in Bollgard II plots compared with Bollgard but means did not differ significantly (P > 0.05). Single and dual-toxin genotypes remained superior (P < 0.05) compared with conventional cotton against the tobacco budworm, Heliothis virescens (F.). The addition of Cry2Ab had no significant (P > 0.05) impact on Cry1Ac expression in Bollgard II compared with Cry1Ac expression in Bollgard. Furthermore, throughout the season Cry2Ab was present at much higher levels in the plant compared with Cry1Ac for Bollgard II plants. Possible species-specific reasons for increased efficacy of Bollgard II over Bollgard are discussed.

  16. 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.

  17. Ultrasensitive detection of endotoxins using computationally designed nanoMIPs.

    PubMed

    Altintas, Zeynep; Abdin, Mohammed J; Tothill, Alexander M; Karim, Kal; Tothill, Ibtisam E

    2016-09-01

    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. PMID:27543033

  18. 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. PMID:27268595

  19. Pavlovian conditioning of endotoxin-tolerance in rats.

    PubMed

    Oberbeck, Reiner; Kromm, Alexander; Exton, Michael S; Schade, Ullrich; Schedlowski, Manfred

    2003-02-01

    The most fascinating example of the bi-directional interaction between the central nervous system (CNS) and immune system is the behavioral conditioning of immune functions. We therefore investigated the behavioral conditioning of lipopolysaccharide (LPS)-induced endotoxin tolerance using the taste aversion paradigm. The conditioned stimulus (CS) saccharin was paired with the unconditioned stimulus (UCS) LPS over a five (CONDl) or four (COND2) days learning trial. Controls received drinking water with (SHAM) or without (UNT) LPS. Endotoxin tolerance was tested by determination of LPS-induced tumor necrosis factor (TNF)-alpha release. After the avoidance of the induced endotoxin-tolerance the CS saccharin was re-presented in all experimental groups. A the end of the re-exposure period a complete endotoxin tolerance was noticed in the CONDl- and COND2-group. In contrast, no effect of saccharin administration was observed in the SHAM- or UNT-group. Our data demonstrate for the first time the behavioral conditioning of endotoxin tolerance. Furthermore, these results contribute new aspects to the mechanisms underlying the development and modulation of endotoxin tolerance.

  20. Biopanning of endotoxin-specific phage displayed peptides.

    PubMed

    Thomas, Celestine J; Sharma, Shilpi; Kumar, Gyanendra; Visweswariah, Sandhya S; Surolia, Avadhesha

    2003-07-18

    Systemic bacterial infections frequently lead to a plethora of symptoms termed "endotoxic shock" or "sepsis." Characterized by hypotension, coagulation abnormalities, and multiple organ failure, treatment of sepsis still remains mostly supportive. Of the various experimental therapeutic interventional strategies, neutralization of endotoxin by peptides or proteins is becoming popular recently. Hence, design of endotoxin binding peptides is gaining currency as their structural complexity and mode of recognition of endotoxin precludes mounting of resistance against them by the susceptible bacteria by genetic recombination, mutation, etc. Earlier work from our laboratory had shown that the amphiphilic cationic peptides are good ligands for endotoxin binding. In this study, we report the results of studies with the 12 selected lipid A binding phage displayed peptides by biopanning of a repertoire of a random pentadecapeptide library displayed on the filamentous M-13 phage. A comparison of the sequences revealed no consensus sequence between the 12 selected peptides suggesting that the lipid A binding motif is not sequence specific which is in accord with the sequence variation seen with the naturally occurring anti-microbial and/or endotoxin binding peptides. Thus, the flexibility of the peptides coupled with their plasticity in recognizing the lipid A moiety, explains their tight binding to endotoxin. At a structural level, asymmetric distribution of the charged polar residues on one face of the helix and non-polar residues on the opposite face appears to correlate with their activity.

  1. 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.

  2. 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.

  3. 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.

  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. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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...

  10. Endotoxin inactivation in water by using medium-pressure UV lamps.

    PubMed

    Anderson, W B; Huck, P M; Dixon, D G; Mayfield, C I

    2003-05-01

    Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be approximately 0.55 endotoxin unit/ml per mJ/cm(2) of irradiation delivered. PMID:12732578

  11. 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.

  12. Fan deltas and braid deltas: conceptual problems

    SciTech Connect

    McPherson, J.G.; Shanmugam, G.; Moiola, R.J.

    1986-05-01

    The concept of fan deltas has been widely misinterpreted in the geologic literature. A true fan delta is defined as an alluvial fan deposited into a standing body of water. Such sequences are of limited areal extent and are, as expected, uncommon in the rock record. By contrast, braid deltas (herein defined), formed by progradation of a braided fluvial system into a standing body of water, are a common geomorphic feature in many modern settings, and their deposits are common in the geologic record. Braid-delta sequences are often identified as fan deltas, on the false premise that coarse-grained deposits in a deltaic setting are always part of an alluvial fan complex. The authors find that most published examples of so called fan deltas contain no direct evidence for the presence of an alluvial fan. Even in examples where an alluvial fan could be documented, the authors found that, in many cases, the alluvial fan complex was far removed from the shoreline, separated by an extensive braid plain. The authors suggest that such systems are better classified as braid deltas. They consider that it is essential to distinguish the environmental setting of true fan deltas from that of braid deltas. Misclassification will lead to incorrect interpretations of expected facies, sandstone geometry, reservoir quality, and tectonic settings. Criteria based on geometry, vertical and lateral lithofacies associations, and paleocurrent patterns should be used to correctly identify and distinguish these depositional systems.

  13. 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.

  14. Harnessing aptamers for electrochemical detection of endotoxin.

    PubMed

    Kim, Sung-Eun; Su, Wenqiong; Cho, MiSuk; Lee, Youngkwan; Choe, Woo-Seok

    2012-05-01

    Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K(d)) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay. PMID:22370280

  15. The macrophage mediates the renoprotective effects of endotoxin preconditioning.

    PubMed

    Hato, Takashi; Winfree, Seth; Kalakeche, Rabih; Dube, Shataakshi; Kumar, Rakesh; Yoshimoto, Momoko; Plotkin, Zoya; Dagher, Pierre C

    2015-06-01

    Preconditioning is a preventative approach, whereby minimized insults generate protection against subsequent larger exposures to the same or even different insults. In immune cells, endotoxin preconditioning downregulates the inflammatory response and yet, preserves the ability to contain infections. However, the protective mechanisms of preconditioning at the tissue level in organs such as the kidney remain poorly understood. Here, we show that endotoxin preconditioning confers renal epithelial protection in various models of sepsis in vivo. We also tested the hypothesis that this protection results from direct interactions between the preconditioning dose of endotoxin and the renal tubules. This hypothesis is on the basis of our previous findings that endotoxin toxicity to nonpreconditioned renal tubules was direct and independent of immune cells. Notably, we found that tubular protection after preconditioning has an absolute requirement for CD14-expressing myeloid cells and particularly, macrophages. Additionally, an intact macrophage CD14-TRIF signaling pathway was essential for tubular protection. The preconditioned state was characterized by increased macrophage number and trafficking within the kidney as well as clustering of macrophages around S1 proximal tubules. These macrophages exhibited increased M2 polarization and upregulation of redox and iron-handling molecules. In renal tubules, preconditioning prevented peroxisomal damage and abolished oxidative stress and injury to S2 and S3 tubules. In summary, these data suggest that macrophages are essential mediators of endotoxin preconditioning and required for renal tissue protection. Preconditioning is, therefore, an attractive model to investigate novel protective pathways for the prevention and treatment of sepsis.

  16. 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.

  17. Endotoxin neutralization as a biomonitor for inflammatory bowel disease.

    PubMed

    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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Endotoxin Exposure and Eczema in the First Year of Life

    PubMed Central

    Phipatanakul, Wanda; Celedón, Juan C.; Raby, Benjamin A.; Litonjua, Augusto A.; Milton, Donald K.; Sredl, Diane; Weiss, Scott T.; Gold, Diane R.

    2005-01-01

    Objective Exposure to endotoxin in early life has been proposed as a factor that may protect against the development of allergic diseases such as eczema. The objective of this study was to examine the relation between endotoxin exposure in early life and eczema in the first year of life in children with parental history of asthma or allergies. Methods This study used a prospective birth cohort study of 498 children who had a history of allergy or asthma in at least 1 parent and lived in metropolitan Boston. A subset of 401 living rooms had house dust samples adequate for analysis of endotoxin. Results In multivariate analyses adjusting for gender, income, and season of birth, endotoxin levels in the living room at 2 to 3 months of age was inversely associated with physician- or nurse-diagnosed eczema in the first year of life (odds ratio [OR] for each quartile increment: 0.76; 95% confidence interval [CI]: 0.61–0.96). Exposure to a dog in the home at age 2 to 3 months was also inversely associated with eczema in the first year of life, but the CI widened when endotoxin was included in the multivariate model (OR: 0.54; 95% CI: 0.27–1.09). Other variables associated with eczema in the first year of life included paternal history of eczema (OR: 1.91; 95% CI: 1.03–3.55) and maternal specific immunoglobulin E positivity to ≥1 allergen (OR: 1.61; 95% CI: 1.01–2.56). Conclusions Among children with parental history of asthma or allergies, exposure to high levels of endotoxin in early life may be protective against eczema in the first year of life. In these children, paternal history of eczema and maternal sensitization to at least 1 allergen are associated with an increased risk of eczema in the first year of life. PMID:15231902

  3. Coarse particulate matter and airborne endotoxin within wood stove homes.

    PubMed

    McNamara, M; Thornburg, J; Semmens, E; Ward, T; Noonan, C

    2013-12-01

    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 (PM₁₀₋₂.₅) 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.

  4. Imaging Phenotype of Occupational Endotoxin-Related Lung Function Decline

    PubMed Central

    Lai, Peggy S.; Hang, Jing-qing; Zhang, Feng-ying; Sun, J.; Zheng, Bu-Yong; Su, Li; Washko, George R.; Christiani, David C.

    2016-01-01

    Background: 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. Objective: We identified the relative contributions of smoking and occupational endotoxin exposure to parenchymal and airway remodeling as defined by quantitative computed tomography (CT). Methods: 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. Results: 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. Conclusions: 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

  5. Endotoxin impairs the human pacemaker current If.

    PubMed

    Zorn-Pauly, Klaus; Pelzmann, Brigitte; Lang, Petra; Mächler, Heinrich; Schmidt, Hendrik; Ebelt, Henning; Werdan, Karl; Koidl, Bernd; Müller-Werdan, Ursula

    2007-12-01

    LPSs trigger the development of sepsis by gram-negative bacteria and cause a variety of biological effects on host cells, including alterations on ionic channels. Because heart rate variability is reduced in human sepsis and endotoxemia, we hypothesized that LPS affects the pacemaker current I(f) in human heart, which might--at least in part--explain this phenomenon. Isolated human myocytes from right atrial appendages were incubated for 6 to 10 h with LPS (1 and 10 microg/mL) and afterwards used to investigate the pacemaker current I(f). I(f) was measured with the whole-cell patch-clamp technique (at 37 degrees C). Incubation of atrial myocytes with 10 microg/mL LPS was found to significantly impair I(f) by suppressing the current at membrane potentials positive to -80 mV and slowing down current activation, but without effecting maximal current conductance. Furthermore, in incubated cells (10 microg/mL), the response of I(f) to [beta]-adrenergic stimulation (1 microM isoproterenol) was significantly larger compared with control cells (shift of half-maximal activation voltage to more positive potentials amounted to -10 and -14 mV in untreated and treated cells, respectively). Simulations using a spontaneously active sinoatrial cell model demonstrated that LPS-induced I(f) impairment reduced the responsiveness of the model cell to fluctuations of autonomic input. This study showed a direct impact of LPS on the cardiac pacemaker current I(f). The LPS-induced I(f) impairment may contribute to the clinically observed reduction in heart rate variability under septic conditions and in cardiac diseases such as heart failure, where endotoxin can be of pathophysiological relevance.

  6. Metal ions potentiate microglia responsiveness to endotoxin.

    PubMed

    Rachmawati, Dessy; Peferoen, Laura A N; Vogel, Daphne Y S; Alsalem, Inás W A; Amor, Sandra; Bontkes, Hetty J; von Blomberg, B Mary E; Scheper, Rik J; van Hoogstraten, Ingrid M W

    2016-02-15

    Oral metal exposure has been associated with diverse adverse reactions, including neurotoxicity. We showed previously that dentally applied metals activate dendritic cells (MoDC) via TLR4 (Ni, Co, Pd) and TLR3 (Au). It is still unknown whether the low levels of dental metals reaching the brain can trigger local innate cells or prime them to become more responsive. Here we tested whether dentally applied metals (Cr, Fe, Co, Ni, Cu, Zn, Au, Hg) activate primary human microglia in vitro and, as a model, monocytic THP-1-cells, in high non-toxic as well as near-physiological concentrations. In addition the effects of 'near-physiological' metal exposure on endotoxin (LPS) responsiveness of these cells were evaluated. IL-8 and IL-6 production after 24h was used as read out. In high, non-toxic concentrations all transition metals except Cr induced IL-8 and IL-6 production in microglia, with Ni and Co providing the strongest stimulation. When using near-physiological doses (up to 10× the normal plasma concentration), only Zn and Cu induced significant IL-8 production. Of note, the latter metals also markedly potentiated LPS responsiveness of microglia and THP-1 cells. In conclusion, transition metals activate microglia similar to MoDCs. In near-physiological concentrations Zn and Cu are the most effective mediators of innate immune activation. A clear synergism between innate responses to Zn/Cu and LPS was observed, shedding new light on the possible relation between oral metal exposure and neurotoxicity.

  7. 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.

  8. 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.

  9. [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.

  10. 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.

  11. 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.

  12. 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.

  13. Optimizing the extraction, storage, and analysis of airborne endotoxins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While the Limulus amebocyte lysate (LAL) assay is part of most procedures to assess airborne endotoxin exposure, there is no universally agreed upon standard procedure. The purpose of this study was to fill in additional knowledge gaps with respect to the extraction, storage, and analysis of endotox...

  14. 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...

  15. 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.

  16. 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.

  17. [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.

  18. 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.

  19. [Endotoxin Is a Companent in Pathogenesis of Chronic Viral Diseases].

    PubMed

    Anikhovskaya, I; Kubatiev, A; Khasanova, G; Yakovlev, M

    2015-01-01

    The level of endotoxin and indicators of activity of antiendotoxin immunity (antibody concentration to glycolipid Re-chemotype and general antigen of enterobacteria) were estimated in serum of 174 patients with persistent viral infections (viruses: herpes simplex, hepatitis C, human immunodeficiency). The presence of markers of systemic inflammatory response syndrome (interleukin IL-1β) and acquired immunodeficiency (CD4+) in HIV-infected patients were also determined. Persistent viral infections are accompanied by endotoxin aggression intestinal origin (caused by them), which is able to induce the development of systemic inflammatory response syndrome. In HIV-infected patients with this syndrome is cyclical, when the phase of hyperactivity replaced immunodeficiency. Schematically, this process can be represented as the following sequence of events: HIV-mediated damage to the intestinal barrier--the development of endotoxin aggression--induction ofsystemic inflammatory response syndrome--the depletion of the immune system, which is transient and is related to the duration of activity of the virus replication cycle, i.e., with damage to enterocytes. Using antiendotoxin component (means of reducing levels of endotoxin in the blood) in the scheme of treatment of persistent viral infections can serve as an element of a successful prevention of complications. PMID:26237956

  20. Endotoxin recovery using limulus amebocyte lysate (LAL) assay.

    PubMed

    Bolden, Jay S; Warburton, Rob E; Phelan, Robert; Murphy, Marie; Smith, Kelly R; De Felippis, Michael R; Chen, Dayue

    2016-09-01

    A phenomenon initially reported by Chen and Vinther in 2013 [1], and now commonly referred to as low endotoxin recovery (LER), has prompted the Food and Drug Administration (FDA) to request specific data demonstrating the capability of the LAL BET method (i.e., USP <85>) to recover endotoxin from spiked samples over time. The results of these spike/hold recovery studies are expected to be included in the Biologics License Applications (BLA) for review by the Center for Drug Evaluation and Research (CDER) Hughes (2014) and Hughes et al. (2015) [2,3]. Such studies involve spiking a known amount of a surrogate endotoxin, such as purified lipopolysaccharide (LPS), into undiluted biological products and then testing at different time points to determine the recovery over time. We report here the experience and learning gained from conducting spike/hold recovery studies for a monoclonal antibody (Mab) product. Results from initial hold studies spiked with purified LPS showed rapid loss of endotoxin activity in the drug substance (DS) and significant batch-to-batch variation in the drug product (DP). After careful review and examination of the experimental details, it was determined that the study design and execution differed from the routine batch release USP <85> BET method with regard to mixing time and sampling scheme. The hold study design was subsequently revised so that the mixing time and sampling were the same as the verified USP <85> BET method used for routine batch release testing. The spike/hold recovery studies were repeated and the results demonstrated that LPS could be consistently recovered over time. These findings highlight the importance of carefully controlling sample preparation procedures in a spike/hold recovery study in order to demonstrate the suitability of using the LAL BET method for endotoxin detection. PMID:27470947

  1. Characterization of an aerosol chamber for human exposures to endotoxin.

    PubMed

    Taylor, L; Reist, P C; Boehlecke, B A; Jacobs, R R

    2000-03-01

    The objective of this study was to develop and characterize an exposure chamber in which human subjects could be exposed to low dust concentrations carrying an endotoxin coating. An exposure chamber, dust dispersion method, and endotoxin characterization technique were developed for inhalation exposures. A 6.27 m3 exposure chamber was designed and constructed from cinder block, glass windows, and Plexiglas. Using an acetone adhesion process, Enterobacter agglomerans were adsorbed onto respirable cellulose particles to create the endotoxin aerosol. The size distribution of the endotoxin-treated particles was verified using light microscopy and cascade impactors. A dry powder dust generator was refined to consistently disperse small quantities of the aerosol into the chamber to maintain dust concentrations at approximately 250 micrograms/m3. Dust levels during the chamber exposures were monitored using a portable continuous aerosol monitor (PCAM). During initial exposure runs, PCAM monitoring stations were positioned at different locations within a 0.5-meter matrix to document mixing patterns. Total dust and cascade impactor samples were collected throughout each exposure period to characterize the chamber operating system and insure the mean airborne dust concentration fulfilled target levels. A one-factor analysis of variance at the 95 percent confidence interval illustrated that there was not a statistically significant difference in the mean dust concentration throughout the exposure runs compared to the individual runs. Together the consistency of the total dust filters, endotoxin concentrations, and aerosol-monitoring instrument were adequate to allow use of the chamber for experimental studies involving human volunteers.

  2. 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

    2013-09-01

    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. PMID:23927534

  3. 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.

  4. 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

    2013-09-01

    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.

  5. Endotoxin levels in homes and classrooms of Dutch school children and respiratory health.

    PubMed

    Jacobs, José H; Krop, Esmeralda J M; de Wind, Siegfried; Spithoven, Jack; Heederik, Dick J J

    2013-08-01

    Several studies describe indoor pollutant exposure in homes and to a lesser extent in schools. Population studies that include both environments are sparse. This study aims to assess endotoxin levels in primary schools and homes of children. Endotoxin was also studied in relation to asthma and sensitisation. 10 schools with (index) and without (reference) dampness were selected, based on reports and inspections. Cases and controls were selected from 169 homes based on the presence or absence of asthma-like symptoms of children. Classroom and bedroom airborne settled dust was sampled using electrostatic dust fall collectors. Average endotoxin levels in schools ranged from 2178 to 6914 endotoxin units (EU)·m(-2) per week compared with 462-1285 EU·m(-2) per week in homes. After mutual adjustment for home and school endotoxin, school endotoxin was positively associated with nonatopic asthma (OR 1.11, 95% CI 0.97-1.27), while no associations with endotoxin were found at home. The high endotoxin levels in schools compared with homes indicate that exposure at school can contribute considerably to environmental endotoxin exposure of children and teachers. Our results also suggest that endotoxin in schools may be associated with nonatopic asthmatic symptoms in pupils, although the results require reproduction because of the modest sample size.

  6. 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.

  7. 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.

  8. Delta launcher enhanced

    NASA Astrophysics Data System (ADS)

    1987-08-01

    The next-generation, 'Delta II' version of the Delta expendable launch vehicle will be able to launch over 4000 lbs into geosynchronous transfer orbit (GTO), as required by the USAF's Navstar GPS; the current Delta 3920 configuration can loft only 2800 lbs into GEO. Three distinct growth configurations of the Delta II are planned: the 6925, whose booster propellant tanks will be extended by 12 ft; the 7925, whose improved booster engine will increase nozzle expansion ratio from 8:1 to 12:1; and the 'enhanced ' Delta II, with stretched graphite-epoxy solid rocket motor cases. In this final form, Delta II will boost 4010 lbs into GTO, or 11,110 lbs into LEO.

  9. Exposure to airborne microorganisms and endotoxin in herb processing plants.

    PubMed

    Dutkiewicz, J; Krysińska-Traczyk, E; Skórska, C; Sitkowska, J; Prazmo, Z; Golec, M

    2001-01-01

    Microbiological air sampling was performed in two herb processing plants located in eastern Poland. Air samples for determination of the levels of bacteria, fungi, dust and endotoxin were collected at 14 sites during cleaning, cutting, grinding, sieving, sorting and packing of 11 kinds of herbs (nettle, caraway, birch, celandine, marjoram, mint, peppermint, sage, St. John's wort, calamus, yarrow), used for production of medications, cosmetics and spices. It was found that processing of herbs was associated with a very high pollution of the air with bacteria, fungi, dust and endotoxin. The numbers of microorganisms (bacteria and fungi) in the air of herb processing plants ranged within 40.6-627.4 x 10(3) cfu/m3 (mean +/- S.D = 231.4 +/- 181.0 x 10(3) cfu/m3). The greatest concentrations were noted at the initial stages of production cycle, during cleaning, cutting and grinding of herbs. The numbers of airborne microorganisms were also significantly (p<0.0001) related to the kind of processed herb, being the greatest at processing marjoram, nettle, yarrow and mint. The values of the respirable fraction of airborne microflora in the examined facilities varied within a fairly wide range and were between 14.7-67.7%. The dominant microorganisms in the air of herb processing plants were mesophilic bacteria, among which endospore-forming bacilli (Bacillus spp.) and actinomycetes of the species Streptomyces albus were most numerous. Among Gram-negative bacteria, the most common was endotoxin-producing species Alcaligenes faecalis. Altogether, 37 species or genera of bacteria and 23 species or genera of fungi were identified in the air of herb processing plants, of these, 11 and 10 species or genera respectively were reported as having allergenic and/or immunotoxic properties. The concentrations of dust and bacterial endotoxin in the air of herb processing plants were large with extremely high levels at some sampling sites. The concentrations of airborne dust ranged within 3

  10. Exposure to airborne microorganisms and endotoxin in herb processing plants.

    PubMed

    Dutkiewicz, J; Krysińska-Traczyk, E; Skórska, C; Sitkowska, J; Prazmo, Z; Golec, M

    2001-01-01

    Microbiological air sampling was performed in two herb processing plants located in eastern Poland. Air samples for determination of the levels of bacteria, fungi, dust and endotoxin were collected at 14 sites during cleaning, cutting, grinding, sieving, sorting and packing of 11 kinds of herbs (nettle, caraway, birch, celandine, marjoram, mint, peppermint, sage, St. John's wort, calamus, yarrow), used for production of medications, cosmetics and spices. It was found that processing of herbs was associated with a very high pollution of the air with bacteria, fungi, dust and endotoxin. The numbers of microorganisms (bacteria and fungi) in the air of herb processing plants ranged within 40.6-627.4 x 10(3) cfu/m3 (mean +/- S.D = 231.4 +/- 181.0 x 10(3) cfu/m3). The greatest concentrations were noted at the initial stages of production cycle, during cleaning, cutting and grinding of herbs. The numbers of airborne microorganisms were also significantly (p<0.0001) related to the kind of processed herb, being the greatest at processing marjoram, nettle, yarrow and mint. The values of the respirable fraction of airborne microflora in the examined facilities varied within a fairly wide range and were between 14.7-67.7%. The dominant microorganisms in the air of herb processing plants were mesophilic bacteria, among which endospore-forming bacilli (Bacillus spp.) and actinomycetes of the species Streptomyces albus were most numerous. Among Gram-negative bacteria, the most common was endotoxin-producing species Alcaligenes faecalis. Altogether, 37 species or genera of bacteria and 23 species or genera of fungi were identified in the air of herb processing plants, of these, 11 and 10 species or genera respectively were reported as having allergenic and/or immunotoxic properties. The concentrations of dust and bacterial endotoxin in the air of herb processing plants were large with extremely high levels at some sampling sites. The concentrations of airborne dust ranged within 3

  11. Removing Endotoxin from Metallic Biomaterials with Compressed Carbon Dioxide-Based Mixtures

    PubMed Central

    Tarafa, Pedro J.; Williams, Eve; Panvelker, Samir; Zhang, Jian; Matthews, Michael A.

    2010-01-01

    Bacterial endotoxins have strong affinity for metallic biomaterials because of surface energy effects. Conventional depyrogenation methods may not eradicate endotoxins and may compromise biological properties and functionality of metallic instruments and implants. We evaluated the solubilization and removal of E. coli endotoxin from smooth and porous titanium (Ti) surfaces and stainless steel lumens using compressed CO2-based mixtures having water and/or surfactant Ls-54. The CO2/water/Ls-54 ternary mixture in the liquid CO2 region (25 °C and 27.6 MPa) with strong mixing removed endotoxin below detection levels. This suggests that the ternary mixture penetrates and dissolves endotoxins from all the tested substrates. The successful removal of endotoxins from metallic biomaterials with compressed CO2 is a promising cleaning technology for biomaterials and reusable medical devices. PMID:21499532

  12. Removal of endotoxin from protein solutions by phase separation using Triton X-114.

    PubMed

    Aida, Y; Pabst, M J

    1990-09-14

    Endotoxin contamination of protein solutions was reduced by a phase separation technique using the detergent, Triton X-114. Protein solutions containing endotoxin were treated with Triton X-114 on ice. The solution was then warmed to 37 degrees C, whereupon two phases formed. The Triton X-114 phase, containing the endotoxin, was precipitated by centrifugation. The first cycle of phase separation produced a 1000-fold reduction of endotoxin from contaminated preparations of cytochrome c, catalase and albumin. Complete removal of endotoxin could be achieved by further cycles of phase separation. Each cycle of phase separation resulted in only a 2% loss of protein, and could be completed within 15 min. The small amount of detergent (0.018%) that persisted in protein solution could be removed by gel filtration or absorption. Proteins treated by this procedure retained normal functions. This phase separation technique provides a rapid and gentle method for removing endotoxin from protein solutions.

  13. 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

    2016-04-01

    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.

  14. 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.

  15. 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

  16. School Endotoxin Exposure and Asthma Morbidity in Inner-city Children

    PubMed Central

    Lai, Peggy S.; Sheehan, William J.; Gaffin, Jonathan M.; Petty, Carter R.; Coull, Brent A.; Gold, Diane R.; Phipatanakul, Wanda

    2015-01-01

    BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01756391; URL: www.clinicaltrials.gov PMID:26087201

  17. Indoor Pollutant Exposures Modify the Effect of Airborne Endotoxin on Asthma in Urban Children

    PubMed Central

    Hansel, Nadia N.; Aloe, Charles; Schiltz, Allison M.; Peng, Roger D.; Rabinovitch, Nathan; Ong, Mary Jane; Williams, D’Ann L.; Breysse, Patrick N.; Diette, Gregory B.; Liu, Andrew H.

    2013-01-01

    Rationale: The effect of endotoxin on asthma morbidity in urban populations is unclear. Objectives: To determine if indoor pollutant exposure modifies the relationships between indoor airborne endotoxin and asthma health and morbidity. Methods: One hundred forty-six children and adolescents with persistent asthma underwent repeated clinical assessments at 0, 3, 6, 9, and 12 months. Home visits were conducted at the same time points for assessment of airborne nicotine, endotoxin, and nitrogen dioxide (NO2) concentrations. The effect of concomitant pollutant exposure on relationships between endotoxin and asthma outcomes were examined in stratified analyses and statistical models with interaction terms. Measurements and Main Results: Both air nicotine and NO2 concentrations modified the relationships between airborne endotoxin and asthma outcomes. Among children living in homes with no detectable air nicotine, higher endotoxin was inversely associated with acute visits and oral corticosteroid bursts, whereas among those in homes with detectable air nicotine, endotoxin was positively associated with these outcomes (interaction P value = 0.004 and 0.07, respectively). Among children living in homes with lower NO2 concentrations (<20 ppb), higher endotoxin was positively associated with acute visits, whereas among those living in homes with higher NO2 concentrations, endotoxin was negatively associated with acute visit (interaction P value = 0.05). NO2 also modified the effect of endotoxin on asthma symptom outcomes in a similar manner. Conclusions: The effects of household airborne endotoxin exposure on asthma are modified by coexposure to air nicotine and NO2, and these pollutants have opposite effects on the relationships between endotoxin and asthma-related outcomes. PMID:24066676

  18. 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.

  19. 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.

  20. Within-Home versus Between-Home Variability of House Dust Endotoxin in a Birth Cohort

    PubMed Central

    Abraham, Joseph H.; Gold, Diane R.; Dockery, Douglas W.; Ryan, Louise; Park, Ju-Hyeong; Milton, Donald K.

    2005-01-01

    Endotoxin exposure has been proposed as an environmental determinant of allergen responses in children. To better understand the implications of using a single measurement of house dust endotoxin to characterize exposure in the first year of life, we evaluated room-specific within-home and between-home variability in dust endotoxin obtained from 470 households in Boston, Massachusetts. Homes were sampled up to two times over 5–11 months. We analyzed 1,287 dust samples from the kitchen, family room, and baby’s bedroom for endotoxin. We fit a mixed-effects model to estimate mean levels and the variation of endotoxin between homes, between rooms, and between sampling times. Endotoxin ranged from 2 to 1,945 units per milligram of dust. Levels were highest during summer and lowest in the winter. Mean endotoxin levels varied significantly from room to room. Cross-sectionally, endotoxin was moderately correlated between family room and bedroom floor (r = 0.30), between family room and kitchen (r = 0.32), and between kitchen and bedroom (r = 0.42). Adjusting for season, the correlation of endotoxin levels within homes over time was 0.65 for both the bedroom and kitchen and 0.54 for the family room. The temporal within-home variance of endotoxin was lowest for bedroom floor samples and highest for kitchen samples. Between-home variance was lowest in the family room and highest for kitchen samples. Adjusting for season, within-home variation was less than between-home variation for all three rooms. These results suggest that room-to-room and home-to-home differences in endotoxin influence the total variability more than factors affecting endotoxin levels within a room over time. PMID:16263505

  1. 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

  2. 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.

  3. Masking of endotoxin in surfactant samples: Effects on Limulus-based detection systems.

    PubMed

    Reich, Johannes; Lang, Pierre; Grallert, Holger; Motschmann, Hubert

    2016-09-01

    Over the last few decades Limulus Amebocyte Lysate (LAL) has been the most sensitive method for the detection of endotoxins (Lipopolysaccharides) and is well accepted in a broad field of applications. Recently, Low Endotoxin Recovery (LER) in biopharmaceutical drug products has been noticed, whereby the detection of potential endotoxin contaminations is not ensured. Notably, most of these drug products contain surfactants, which can have crucial effects on the detectability of endotoxin. In order to analyze the driving forces of LER, endotoxin detection in samples containing nonionic surfactants in various buffer systems was investigated. The results show that the process of LER is kinetically controlled and temperature-dependent. Furthermore, only the simultaneous presence of nonionic surfactants and components capable of forming metal complexes resulted in LER. In addition, capacity experiments show that even hazardous amounts of endotoxin can remain undetectable within such formulation compositions. In conclusion, the LER phenomenon is caused by endotoxin masking and not by test interference. In this process, the supramolecular structure of endotoxin is altered and exhibits only a limited susceptibility in binding to the Factor C of Limulus-based detection systems. We propose a two-step mechanism of endotoxin masking by complex forming agents and nonionic surfactants. PMID:27464990

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. [Cytokine release after administration of endotoxin containing vaccines

    PubMed

    Ecker, Martina; Müller, Günter

    1998-01-01

    Endotoxins from gram negative bacteria are known to be potent inducers for the synthesis and the release of cytokines such as tumour necrosis factor (TNF) and interleukin 6 (IL-6). The amount of these proinflammatory mediators in plasma from animals and human patients suffering of an acute infection or sepsis, however, is well correlated with the outcome and the prognosis of such diseases (Hack et al., 1989; Ostermann et al., 1997; Rigato, 1996). In connection with regular testing of vaccine lots we determine the release and the kinetic of TNF and IL-6 in piglets after immunisation with different vaccines containing endotoxin. The current results suggest that the amounts of both cytokines increased with elevated endotoxin concentration given with the doses. TNF peaked in plasma after one hour, IL-6 peaked between two and four hours p.appl. We did not find any influence of the gender of the animals. In contrast, the body weight seems to affect the cytokine release in different ways. Determination of cytokine changes in plasma is a sensitive tool for the evaluation of systemic reactions and supports data about the clinical and haematological signs.

  11. 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.

  12. Myocardial failure with altered response to adrenaline in endotoxin shock

    PubMed Central

    Archer, L.T.; Black, M.R.; Hinshaw, L.B.

    1975-01-01

    1 There is a growing concensus that myocardial performance in the early stages of experimental endotoxic and septic shock is relatively normal; however, recent reports have identified an intermediate phase of shock when myocardial dysfunction is clearly apparent. 2 The mechanism of dysfunction has become a subject of intense investigation. A current view is that altered myocardial responsiveness to circulating catecholamines may play an important role in the dysfunction observed after endotoxin administration. The present studies, in which an isolated working heart preparation of the dog was used, were designed to test this hypothesis. This particular experimental preparation was selected to provide an adequate interpretation of results; cardiac output, afterload, and concentrations of adrenaline reaching the coronary vascular bed were controlled in all experiments. Responses to infusions of adrenaline were recorded in the `steady-state' condition. Control (non-shocked) heart responses to adrenaline were highly reproducible in terms of inotropic, chronotropic and coronary vascular behaviour. 3 Results from the study document myocardial dysfunction within 4-6 h following an LD70 endotoxin administration on the basis of increased left ventricular end diastolic pressure (LVEDP), decreased cardiac power and myocardial efficiency, and depressed negative and positive dP/dt parameters. 4 Findings suggest significantly altered responsiveness of the myocardium to infused adrenaline at rates of 1, 2, and 5 μg/min with concentrations between 10 and 1 ng/ml blood. LVEDP was elevated while calculated power and efficiency parameters remained significantly below control values during infusion of adrenaline in endotoxin-treated hearts. Depressions of responsiveness were interpreted to occur on the basis of failure to restore positive and negative dP/dt to normal values and depressed coronary blood flow responses during adrenaline administration. Increases in coronary flow were

  13. Airborne Endotoxin from Indoor and Outdoor Environments:Effect of Sample Dilution on the Kinetic Limulus Amebocyte Lysate (LAL) Assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Airborne endotoxin in occupational environments are a potential respiratory hazard to individuals. In this study, total and inhalable airborne endotoxin samples were collected via filtration from inside animal housing units and downwind from agricultural production sites and a wastewater treatment ...

  14. 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. PMID:3787309

  15. Effect of Tris-acetate buffer on endotoxin removal from human-like collagen used biomaterials.

    PubMed

    Zhang, Huizhi; Fan, Daidi; Deng, Jianjun; Zhu, Chenghui; Hui, Junfeng; Ma, Xiaoxuan

    2014-09-01

    Protein preparation, which has active ingredients designated for the use of biomaterials and therapeutical protein, is obtained by genetic engineering, but products of genetic engineering are often contaminated by endotoxins. Because endotoxin is a ubiquitous and potent proinflammatory agent, endotoxin removal or depletion from protein is essential for researching any biomaterials. In this study, we have used Tris-acetate (TA) buffer of neutral pH value to evaluate endotoxins absorbed on the Pierce high-capacity endotoxin removal resin. The effects of TA buffer on pH, ionic strength, incubation time as well as human-like collagen (HLC) concentration on eliminating endotoxins are investigated. In the present experiments, we design an optimal method for TA buffer to remove endotoxin from recombinant collagen and use a chromogenic tachypleus amebocyte lysate (TAL) test kit to measure the endotoxin level of HLC. The present results show that, the endotoxins of HLC is dropped to 8.3EU/ml at 25 mM TA buffer (pH7.8) with 150 mM NaCl when setting incubation time at 6h, and HLC recovery is about 96%. Under this experimental condition, it is proved to exhibit high efficiencies of both endotoxin removal and collagen recovery. The structure of treated HLC was explored by Transmission Electron Microscopy (TEM), demonstrating that the property and structure of HLC treated by TA buffer are maintained. Compared to the most widely used endotoxin removal method, Triton X-114 extraction, using TA buffer can obtain the non-toxic HLC without extra treatment for removing the toxic substances in Triton X-114. In addition, the present study aims at establishing a foundation for further work in laboratory animal science and providing a foundation for medical grade biomaterials.

  16. The Effect of Residual Endotoxin Contamination on the Neuroinflammatory Response to Sterilized Intracortical Microelectrodes.

    PubMed

    Ravikumar, Madhumitha; Hageman, Daniel J; Tomaszewski, William H; Chandra, Gabriella M; Skousen, John L; Capadona, Jeffrey R

    2014-05-01

    A major limitation to the use of microelectrode technologies in both research and clinical applications is our inability to consistently record high quality neural signals. There is increasing evidence that recording instability is linked, in part, to neuroinflammation. A number of factors including extravasated blood products and macrophage released soluble factors are believed to mediate neuroinflammation and the resulting recording instability. However, the roles of other inflammatory stimuli, such as residual endotoxin contamination, are poorly understood. Therefore, to determine the effect of endotoxin contamination we examined the brain tissue response of C57/BL6 mice to non-functional microelectrodes with a range of endotoxin levels. Endotoxin contamination on the sterilized microelectrodes was measured using a limulus amebocyte lysate test following FDA guidelines. Microelectrodes sterilized by autoclave, dry heat, or ethylene oxide gas, resulted in variable levels of residual endotoxins of 0.55 EU/mL, 0.22 EU/mL, and 0.11 EU/mL, respectively. Histological evaluation at two weeks showed a direct correlation between microglia/macrophage activation and endotoxin levels. Interestingly, astrogliosis, neuronal loss, and blood brain barrier dysfunction demonstrated a threshold-dependent response to bacterial endotoxins. However, at sixteen weeks, no histological differences were detected, regardless of initial endotoxin levels. Therefore, our results demonstrate that endotoxin contamination, within the range examined, contributes to initial but not chronic microelectrode associated neuroinflammation. Our results suggest that minimizing residual endotoxins may impact early recording quality. To this end, endotoxins should be considered as a potent stimulant to the neuroinflammatory response to implanted intracortical microelectrodes.

  17. 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.

  18. 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.

  19. 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.

  20. Modeling river delta formation.

    PubMed

    Seybold, Hansjörg; Andrade, José S; Herrmann, Hans J

    2007-10-23

    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. Enhancement of systemic and sputum granulocyte response to inhaled endotoxin in people with the GSTM1 null genotype

    EPA Science Inventory

    To determine if the GSTM1 null genotype is a risk factor for increased inflammatory response to inhaled endotoxin. Methods 35 volunteers who had undergone inhalation challenge with a 20 000 endotoxin unit dose of Clinical Center Reference Endotoxin (CCRE) were genotyped for the G...

  2. Man made deltas

    PubMed Central

    Maselli, Vittorio; Trincardi, Fabio

    2013-01-01

    The review of geochronological and historical data documents that the largest southern European deltas formed almost synchronously during two short intervals of enhanced anthropic pressure on landscapes, respectively during the Roman Empire and the Little Ice Age. These growth phases, that occurred under contrasting climatic regimes, were both followed by generalized delta retreat, driven by two markedly different reasons: after the Romans, the fall of the population and new afforestation let soil erosion in river catchments return to natural background levels; since the industrial revolution, instead, flow regulation through river dams overkill a still increasing sediment production in catchment basins. In this second case, furthermore, the effect of a reduced sediment flux to the coasts is amplified by the sinking of modern deltas, due to land subsidence and sea level rise, that hampers delta outbuilding and increases the vulnerability of coastal zone to marine erosion and flooding. PMID:23722597

  3. Man made deltas.

    PubMed

    Maselli, Vittorio; Trincardi, Fabio

    2013-01-01

    The review of geochronological and historical data documents that the largest southern European deltas formed almost synchronously during two short intervals of enhanced anthropic pressure on landscapes, respectively during the Roman Empire and the Little Ice Age. These growth phases, that occurred under contrasting climatic regimes, were both followed by generalized delta retreat, driven by two markedly different reasons: after the Romans, the fall of the population and new afforestation let soil erosion in river catchments return to natural background levels; since the industrial revolution, instead, flow regulation through river dams overkill a still increasing sediment production in catchment basins. In this second case, furthermore, the effect of a reduced sediment flux to the coasts is amplified by the sinking of modern deltas, due to land subsidence and sea level rise, that hampers delta outbuilding and increases the vulnerability of coastal zone to marine erosion and flooding. PMID:23722597

  4. Electrochemical endotoxin sensors based on TLR4/MD-2 complexes immobilized on gold electrodes.

    PubMed

    Yeo, Tae Yun; Choi, Ji Suk; Lee, Byung Kook; Kim, Beob Soo; Yoon, Hwa In; Lee, Hyeong Yun; Cho, Yong Woo

    2011-10-15

    Even low concentrations of endotoxins can be life-threatening. As such, continuous effort has been directed toward the development of sensitive and specific endotoxin detection systems. In this paper, we report the design and fabrication of a new electrochemical endotoxin sensor based on a human recombinant toll-like receptor 4 (rhTLR4) and myeloid differentiation-2 (MD-2) complex. The rhTLR4/MD-2 complex, which specifically binds to endotoxin, was immobilized on gold electrodes through a self-assembled monolayer (SAM) technique involving the use of dithiobis(succinimidyl undecanoate) (DSU). The surface topography of the electrodes at each fabrication stage was characterized with a nanosurface profiler and atomic force microscope (AFM). The electrochemical signals generated from interactions between the rhTLR4/MD-2 complex and the endotoxin were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A linear relationship between the peak current and endotoxin concentration was obtained in the range of 0.0005 to 5 EU/mL with a correlation coefficient (R(2)) of 0.978. The estimated limit of detection (LOD) was fairly low, 0.0002 EU/mL. The rhTLR4/MD-2 based sensors exhibited no current responses to dipalmitoylphosphatidylcholine (DPPC) bearing two lipid chains, which is structurally similar to endotoxin, indicating the high specificity of the sensors to endotoxin. PMID:21816600

  5. Single session of Nd:YAG laser intracanal irradiation neutralizes endotoxin in dental root dentin.

    PubMed

    Archilla, José R F; Moreira, Maria S N A; Miyagi, Sueli P H; Bombana, Antônio C; Gutknecht, Norbert; Marques, Márcia M

    2012-11-01

    Endotoxins released in the dental root by Gram-negative microorganisms can be neutralized by calcium hydroxide, when this medication is applied inside the root canal for at least seven days. However, several clinical situations demand faster root canal decontamination. Thus, for faster endotoxin neutralization, endodontists are seeking additional treatments. The in vitro study tested whether or not intracanal Nd:YAG laser irradiation would be able to neutralize endotoxin within the human dental root canal in a single session. Twenty-four human teeth with one root were mounted between two chambers. After conventional endodontic treatment, root canals were contaminated with Escherichia coli endotoxin. Then they were irradiated or not (controls) in contact mode with an Nd:YAG laser (1.5 W, 15 Hz, 100 mJ and pulse fluency of 124  J/cm2). The endotoxin activity was measured using the limulus lysate technique and data were statistically compared (p≤0.05). The concentration of active endotoxin measured in the negative control group was significantly lower than that of the positive control group (p=0.04). The concentrations of endotoxin in both irradiated groups were significantly lower than that of the positive control group (p=0.027) and similar to that of negative control group (p=0.20). A single session of intracanal Nd:YAG laser irradiation is able to neutralize endotoxin in the dental root tissues.

  6. New generation ceramic membranes have the potential of removing endotoxins from dialysis water and dialysate.

    PubMed

    Czermak, P; Ebrahimi, M; Catapano, G

    2005-07-01

    Poor water properties, use of concentrated bicarbonate, and biofilm growth in pipes and storage tanks often cause dialysis water and dialysate contamination with bacteria and endotoxins. High-flux dialysis with bicarbonate may favor endotoxin transfer from the dialysate into the blood exposing patients to serious short-and long-term side effects. Ultrafiltration across hydrophobic synthetic membranes effectively removes endotoxins from dialysis water by combined filtration and adsorption. However, repeated sterilization worsens the membrane separation properties,and limits their use. Ceramic membranes are generally more resistant to harsh operating conditions than polymeric membranes, and may represent an alternative for endotoxin removal. Previously, we proved that the ceramic membranes commercially available at that time were not retentive enough to ensure production of endotoxin-free dialysis water. In this paper, we investigated the endotoxin removal capacity of new generation commercial ceramic membranes with nominal molecular weight cut-off down to 1,000. In dead-end filtration, all investigated membranes produced water meeting, the European standards, or close to,when challenged with low endotoxin concentrations, but only one membrane type succeeded at high endotoxin concentrations. In cross-flow filtration, none produced water meeting the European standard. Moreover, sterilization and rinsing procedures altered the separation properties of two out of three membrane types. PMID:16049903

  7. Marine aerosol as a possible source for endotoxins in coastal areas.

    PubMed

    Lang-Yona, Naama; Lehahn, Yoav; Herut, Barak; Burshtein, Noa; Rudich, Yinon

    2014-11-15

    Marine aerosols, that are very common in the highly populated coastal cities and communities, may contain biological constituents. Some of this biological fraction of marine aerosols, such as cyanobacteria and plankton debris, may influence human health by inflammation and allergic reactions when inhaled. In this study we identify and compare sources for endotoxins sampled on filters in an on-shore and more-inland site. Filter analysis included endotoxin content, total bacteria, gram-negative bacteria and cyanobacteria genome concentrations as well as ion content in order to identify possible sources for the endotoxins. Satellite images of chlorophyll-a levels and back trajectory analysis were used to further study the cyanobacteria blooms in the sea, close to the trajectory of the sampled air. The highest endotoxin concentrations found in the shoreline site were during winter (3.23±0.17 EU/m(3)), together with the highest cyanobacteria genome (1065.5 genome/m(3)). The elevated endotoxin concentrations were significantly correlated with cyanobacterial levels scaled to the presence of marine aerosol (r=0.90), as well as to chlorophyll-a (r=0.96). Filters sampled further inland showed lower and non-significant correlation between endotoxin and cyanobacteria (r=0.70, P value=0.19), suggesting decrease in marine-originated endotoxin, with possible contributions from other sources of gram-negative non-cyanobacteria. We conclude that marine cyanobacteria may be a dominant contributor to elevated endotoxin levels in coastal areas.

  8. Physical and biological properties of U. S. standard endotoxin EC after exposure to ionizing radiation

    SciTech Connect

    Csako, G.; Elin, R.J.; Hochstein, H.D.; Tsai, C.M.

    1983-07-01

    Techniques that reduce the toxicity of bacterial endotoxins are useful for studying the relationship between structure and biological activity. We used ionizing radiation to detoxify a highly refined endotoxin preparation. U.S. standard endotoxin EC. Dose-dependent changes occurred by exposure to /sup 60/Co-radiation in the physical properties and biological activities of the endotoxin. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed gradual loss of the polysaccharide components (O-side chain and R-core) from the endotoxin molecules. In contrast, although endotoxin revealed a complex absorption pattern in the UV range, radiation treatment failed to modify that pattern. Dose-related destruction of the primary toxic component, lipid A, was suggested by the results of activity tests: both the pyrogenicity and limulus reactivity of the endotoxin were destroyed by increasing doses of radiation. The results indicate that the detoxification is probably due to multiple effects of the ionizing radiation on bacterial lipopolysaccharides, and the action involves (i) the destruction of polysaccharide moieties and possibly (ii) the alteration of lipid A component of the endotoxin molecule.

  9. Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

    SciTech Connect

    Layman, D.L.; Diedrich, D.L.

    1987-06-01

    Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by /sup 3/H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in /sup 3/H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin.

  10. Relationship between chicken cellular immunity and endotoxin levels in dust from chicken housing environments.

    PubMed

    Roque, Katharine; Shin, Kyung-Min; Jo, Ji-Hoon; Kim, Hyoung-Ah; Heo, Yong

    2015-01-01

    Hazardous biochemical agents in animal husbandry indoor environments are known to promote the occurrence of various illnesses among workers and animals. The relationship between endotoxin levels in dust collected from chicken farms and various immunological markers was investigated. Peripheral blood was obtained from 20 broiler chickens and 20 laying hens from four different chicken farms in Korea. Concentrations of total or respirable dust in the inside the chicken farm buildings were measured using a polyvinyl chloride membrane filter and mini volume sampler. Endotoxin levels in the dust were determined by the Limulus Amebocyte Lysate Kinetic method. Interferon-γ production by peripheral blood mononuclear cells stimulated with concanavalin A was significantly lower in broilers or layers from the farms with higher endotoxin concentrations than the chickens from the farms with lower endotoxin levels. An opposite pattern was observed for plasma cortisol concentrations with higher cortisol levels found in chickens from the farms with higher endotoxin levels. When peripheral lymphocytes were examined, the percentage of CD3(-)Ia(+) B cells was lower in layers from farms with higher endotoxin levels than those from locations with lower endotoxin levels. Overall, these results suggest a probable negative association between dust endotoxin levels and cell-mediated immunity in chickens.

  11. The involvement of an LPS inducible I kappa B kinase in endotoxin tolerance.

    PubMed

    Kohler, N G; Joly, A

    1997-03-27

    When human ovarian carcinoma cells are challenged with endotoxin, an I kappa B kinase is transiently induced within 3 to 5 min. This enzyme activity causes the hyperphosphorylation and subsequent degradation of I kappa B which allows NF-kappa B to translocate to the nucleus where it activates transcription. When endotoxin treated cells are rechallenged with a second dose of LPS, I kappa B kinase is not detected and I kappa B remains in the cytoplasm where it sequesters NF-kappa B. We report here the absence of endotoxin inducible I kappa B kinase activity in endotoxin tolerant cells suggesting that I kappa B kinase may play an important role in endotoxin tolerance. When cells tolerant to endotoxin are treated with TNF, I kappa B kinase activity is induced. Thus cells that are endotoxin tolerant are not cross tolerant to TNF. Dexamethasone, a known inhibitor of NF-kappa B activation does not inhibit endotoxin dependent induction of I kappa B kinase suggesting that the mechanism of action of dexamethasone is different from the tolerance mechanism reported here.

  12. Single session of Nd:YAG laser intracanal irradiation neutralizes endotoxin in dental root dentin

    NASA Astrophysics Data System (ADS)

    Archilla, José R. F.; Moreira, Maria S. N. A.; Miyagi, Sueli P. H.; Bombana, Antônio C.; Gutknecht, Norbert; Marques, Márcia M.

    2012-11-01

    Endotoxins released in the dental root by Gram-negative microorganisms can be neutralized by calcium hydroxide, when this medication is applied inside the root canal for at least seven days. However, several clinical situations demand faster root canal decontamination. Thus, for faster endotoxin neutralization, endodontists are seeking additional treatments. The in vitro study tested whether or not intracanal Nd:YAG laser irradiation would be able to neutralize endotoxin within the human dental root canal in a single session. Twenty-four human teeth with one root were mounted between two chambers. After conventional endodontic treatment, root canals were contaminated with Escherichia coli endotoxin. Then they were irradiated or not (controls) in contact mode with an Nd:YAG laser (1.5 W, 15 Hz, 100 mJ and pulse fluency of 124 J/cm2). The endotoxin activity was measured using the limulus lysate technique and data were statistically compared (p≤0.05). The concentration of active endotoxin measured in the negative control group was significantly lower than that of the positive control group (p=0.04). The concentrations of endotoxin in both irradiated groups were significantly lower than that of the positive control group (p=0.027) and similar to that of negative control group (p=0.20). A single session of intracanal Nd:YAG laser irradiation is able to neutralize endotoxin in the dental root tissues.

  13. Endotoxin detection--from limulus amebocyte lysate to recombinant factor C.

    PubMed

    Ding, Jeak Ling; Ho, Bow

    2010-01-01

    Gram negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. The endotoxin, also known as lipopolysaccharide (LPS), is found in the outer membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines. However, excessive inflammation causes multiple organ failure and death. Endotoxins, which are ubiquitous pathogenic molecules, are a bane to the pharmaceutical industry and healthcare community. Thus early and sensitive detection of endotoxin is crucial to prevent endotoxaemia. The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. In vivo, Factor C is the perfect biosensor, which alerts the horseshoe crab of the presence of a Gram-negative invader. The hemostatic end-point entraps the invader, killing it and limiting further infection. However, as an in vitro endotoxin detection tool, variations in the sensitivity and specificity of LAL to endotoxin, and the dwindling supply of horseshoe crabs are posing increasing challenges to the biotechnology industry. This has necessitated the innovation of an alternative test for endotoxin. Thus, Factor C became the obvious, albeit tricky target for the recombinant technology effort. This chapter documents the backwater of mining the natural blood lysate of the endangered species to the monumental effort of genetic engineering, to produce recombinant Factor C (rFC). The rFC is a 132 kDa molecule, which was produced as a proenzyme inducible by the presence of trace levels of endotoxin. The rFC forms the basis of the "PyroGene" kit, which is a novel micro

  14. Endotoxin and tumor necrosis factor-alpha-induced interleukin-8 release in humans.

    PubMed

    van Deventer, S J; Hart, M; van der Poll, T; Hack, C E; Aarden, L A

    1993-02-01

    Neutrophil recruitment and activation are thought to play an important role in tissue damage observed in septicemia. Interleukin-8 (IL-8) is a small cytokine with important neutrophil-activating and chemoattractant properties. IL-8 release was studied after injection of human volunteers with low doses of either endotoxin (2 ng/kg of body weight) or tumor necrosis factor-alpha (TNF alpha) (50 micrograms/m2). After TNF-alpha injection, IL-8 appeared at 30 min, whereas increased levels were first observed after 90 min in endotoxin-challenged volunteers. Peak levels were measured at 120 min after both endotoxin (192 +/- 193 ng/L) and TNF alpha (500 +/- 236 ng/L) injection. These data indicate that IL-8 is released in humans after injection of endotoxin and TNF alpha and suggest that endotoxin-induced IL-8 release is mediated by TNF alpha.

  15. Abortifacient effects of Vibrio cholerae exo-enterotoxin and endotoxin in mice.

    PubMed

    Gasic, G J; Gasic, T B; Strauss, J F

    1975-11-01

    To study antifertility properties of microbial toxins, exoenterotoxin and endotoxin from Vibrio cholerae were injected intravenously into mice at different times during pregnancy. The two substances induced termination of pregnancy, but the patterns of abortifacient activity were different. Exotoxin terminated pregnancy in mice when administered between Days 4 and 10 of gestation, but abortifacient activity was reduced in animals more than 10 days pregnant; exogenous progesterone did not protect the pregnancies. Endotoxin was most effective in terminating pregnancy when injected after mid-gestation and the active principle was heat-stable; exogenous progesterone was not able to prevent the effects of endotoxin. Animals treated with endotoxin on Day 17 often gave birth to live young prematurely; indomethacin reduced the incidence of premature littering. The results demonstrate that exo- and endotoxins have antifertility properties and both appear to act on intrauterine targets rather than inducing progestin deficiency.

  16. A new method for concentration analysis of bacterial endotoxins in perfluorocarbon

    NASA Astrophysics Data System (ADS)

    Chen, Dan-Dan; Feng, Xiao-Ming; Wang, Chun-Ren; Huang, Qing-Quan; Yang, Zhao-Peng; Meng, Qing-Yuan

    2014-12-01

    This communication demonstrates the feasibility of the gel-clot method for the analysis of bacterial endotoxins in water extracts of perfluorocarbon which is a water insoluble liquid medical device. Perfluorocarbon (10 mL) was shaken with 10mL water for 15 min at 2000 r/min and the endotoxin present was extracted to the aqueous phase without interference inhibition/enhancement of the product and the recovery of endotoxin added to perfluorocarbon was determined. A validation study confirmed that endotoxins presented in perfluorocarbon pass over into the aqueous phase at concentrations of 20, 10 and 5 EU/mL with recoveries from 86.8% to 96.8%. Therefore, the gel-clot test is suitable for detecting bacterial endotoxins in perfluorocarbon which is a water insoluble medical device.

  17. Evaluation of a low-cost electrostatic dust fall collector for indoor air endotoxin exposure assessment.

    PubMed

    Noss, Ilka; Wouters, Inge M; Visser, Maaike; Heederik, Dick J J; Thorne, Peter S; Brunekreef, Bert; Doekes, Gert

    2008-09-01

    Exposure to endotoxin in home environments has become a key issue in asthma and allergy research. Most studies have analyzed floor or mattress dust endotoxin, but its validity as a proxy for airborne exposure is unknown, while active airborne dust sampling is not feasible in large-scale population studies because of logistic and financial limitations. We therefore developed and evaluated a simple passive airborne dust collection method for airborne endotoxin exposure assessment. We explored an electrostatic dust fall collector (EDC), consisting of a 42- by 29.6-cm-sized folder with four electrostatic cloths exposed to the air. The EDC was tested during two 14-day periods in seven nonfarm and nine farm homes and in farm stables. In parallel, active airborne dust sampling was performed with Harvard impactors and floor dust collected by vacuuming, using nylon sampling socks. The endotoxin levels could be measured in all EDC cloth extracts. The levels (in EU/m(2)) between EDCs used simultaneously or in different sampling periods in the same home correlated strongly (r > 0.8). EDC endotoxin also correlated moderately to strongly (r = 0.6 to 0.8) with the endotoxin measured by active airborne dust sampling and living room floor dust sampling and-in farm homes-with the endotoxin captured by the EDC in stables. In contrast, endotoxin levels measured by floor dust sampling showed only a poor correlation with the levels measured by active airborne dust sampling. We therefore conclude that measuring endotoxin levels with the EDC is a valid measure of average airborne endotoxin exposure, while reproducibility over time is at least equivalent to that of reservoir dust analyses.

  18. Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: summary of a symposium.

    PubMed

    Purohit, Vishnudutt; Bode, J Christian; Bode, Christiane; Brenner, David A; Choudhry, Mashkoor A; Hamilton, Frank; Kang, Y James; Keshavarzian, Ali; Rao, Radhakrishna; Sartor, R Balfour; Swanson, Christine; Turner, Jerrold R

    2008-08-01

    This report is a summary of the symposium on Alcohol, Intestinal Bacterial Growth, Intestinal Permeability to Endotoxin, and Medical Consequences, organized by National Institute on Alcohol Abuse and Alcoholism, Office of Dietary Supplements, and National Institute of Diabetes and Digestive and Kidney Diseases of National Institutes of Health in Rockville, Maryland, October 11, 2006. Alcohol exposure can promote the growth of Gram-negative bacteria in the intestine, which may result in accumulation of endotoxin. In addition, alcohol metabolism by Gram-negative bacteria and intestinal epithelial cells can result in accumulation of acetaldehyde, which in turn can increase intestinal permeability to endotoxin by increasing tyrosine phosphorylation of tight junction and adherens junction proteins. Alcohol-induced generation of nitric oxide may also contribute to increased permeability to endotoxin by reacting with tubulin, which may cause damage to microtubule cytoskeleton and subsequent disruption of intestinal barrier function. Increased intestinal permeability can lead to increased transfer of endotoxin from the intestine to the liver and general circulation where endotoxin may trigger inflammatory changes in the liver and other organs. Alcohol may also increase intestinal permeability to peptidoglycan, which can initiate inflammatory response in liver and other organs. In addition, acute alcohol exposure may potentiate the effect of burn injury on intestinal bacterial growth and permeability. Decreasing the number of Gram-negative bacteria in the intestine can result in decreased production of endotoxin as well as acetaldehyde which is expected to decrease intestinal permeability to endotoxin. In addition, intestinal permeability may be preserved by administering epidermal growth factor, l-glutamine, oats supplementation, or zinc, thereby preventing the transfer of endotoxin to the general circulation. Thus reducing the number of intestinal Gram-negative bacteria

  19. Short-term dynamics of indoor and outdoor endotoxin exposure: Case of Santiago, Chile, 2012.

    PubMed

    Barraza, Francisco; Jorquera, Héctor; Heyer, Johanna; Palma, Wilfredo; Edwards, Ana María; Muñoz, Marcelo; Valdivia, Gonzalo; Montoya, Lupita D

    2016-01-01

    Indoor and outdoor endotoxin in PM2.5 was measured for the very first time in Santiago, Chile, in spring 2012. Average endotoxin concentrations were 0.099 and 0.094 [EU/m(3)] for indoor (N=44) and outdoor (N=41) samples, respectively; the indoor-outdoor correlation (log-transformed concentrations) was low: R=-0.06, 95% CI: (-0.35 to 0.24), likely owing to outdoor spatial variability. A linear regression model explained 68% of variability in outdoor endotoxins, using as predictors elemental carbon (a proxy of traffic emissions), chlorine (a tracer of marine air masses reaching the city) and relative humidity (a modulator of surface emissions of dust, vegetation and garbage debris). In this study, for the first time a potential source contribution function (PSCF) was applied to outdoor endotoxin measurements. Wind trajectory analysis identified upwind agricultural sources as contributors to the short-term, outdoor endotoxin variability. Our results confirm an association between combustion particles from traffic and outdoor endotoxin concentrations. For indoor endotoxins, a predictive model was developed but it only explained 44% of endotoxin variability; the significant predictors were tracers of indoor PM2.5 dust (Si, Ca), number of external windows and number of hours with internal doors open. Results suggest that short-term indoor endotoxin variability may be driven by household dust/garbage production and handling. This would explain the modest predictive performance of published models that use answers to household surveys as predictors. One feasible alternative is to increase the sampling period so that household features would arise as significant predictors of long-term airborne endotoxin levels. PMID:27065310

  20. Temporal and Spatial Patterns of Ambient Endotoxin Concentrations in Fresno, California

    PubMed Central

    Tager, Ira B.; Lurmann, Frederick W.; Haight, Thaddeus; Alcorn, Siana; Penfold, Bryan; Hammond, S. Katharine

    2010-01-01

    Background Endotoxins are found in indoor dust generated by human activity and pets, in soil, and adsorbed onto the surfaces of ambient combustion particles. Endotoxin concentrations have been associated with respiratory symptoms and the risk of atopy and asthma in children. Objective We characterized the temporal and spatial variability of ambient endotoxin in Fresno/Clovis, California, located in California’s Central Valley, to identify correlates and potential predictors of ambient endotoxin concentrations in a cohort of children with asthma [Fresno Asthmatic Children’s Environment Study (FACES)]. Methods Between May 2001 and October 2004, daily ambient endotoxin and air pollutants were collected at the central ambient monitoring site of the California Air Resources Board in Fresno and, for shorter time periods, at 10 schools and indoors and outdoors at 84 residences in the community. Analyses were restricted to May–October, the dry months during which endotoxin concentrations are highest. Results Daily endotoxin concentration patterns were determined mainly by meteorologic factors, particularly the degree of air stagnation. Overall concentrations were lowest in areas distant from agricultural activities. Highest concentrations were found in areas immediately downwind from agricultural/pasture land. Among three other measured air pollutants [fine particulate matter, elemental carbon (a marker of traffic in Fresno), and coarse particulate matter (PMc)], PMc was the only pollutant correlated with endotoxin. Endotoxin, however, was the most spatially variable. Conclusions Our data support the need to evaluate the spatial/temporal variability of endotoxin concentrations, rather than relying on a few measurements made at one location, in studies of exposure and and respiratory health effects, particularly in children with asthma and other chronic respiratory diseases. PMID:20494854

  1. Short-term dynamics of indoor and outdoor endotoxin exposure: Case of Santiago, Chile, 2012.

    PubMed

    Barraza, Francisco; Jorquera, Héctor; Heyer, Johanna; Palma, Wilfredo; Edwards, Ana María; Muñoz, Marcelo; Valdivia, Gonzalo; Montoya, Lupita D

    2016-01-01

    Indoor and outdoor endotoxin in PM2.5 was measured for the very first time in Santiago, Chile, in spring 2012. Average endotoxin concentrations were 0.099 and 0.094 [EU/m(3)] for indoor (N=44) and outdoor (N=41) samples, respectively; the indoor-outdoor correlation (log-transformed concentrations) was low: R=-0.06, 95% CI: (-0.35 to 0.24), likely owing to outdoor spatial variability. A linear regression model explained 68% of variability in outdoor endotoxins, using as predictors elemental carbon (a proxy of traffic emissions), chlorine (a tracer of marine air masses reaching the city) and relative humidity (a modulator of surface emissions of dust, vegetation and garbage debris). In this study, for the first time a potential source contribution function (PSCF) was applied to outdoor endotoxin measurements. Wind trajectory analysis identified upwind agricultural sources as contributors to the short-term, outdoor endotoxin variability. Our results confirm an association between combustion particles from traffic and outdoor endotoxin concentrations. For indoor endotoxins, a predictive model was developed but it only explained 44% of endotoxin variability; the significant predictors were tracers of indoor PM2.5 dust (Si, Ca), number of external windows and number of hours with internal doors open. Results suggest that short-term indoor endotoxin variability may be driven by household dust/garbage production and handling. This would explain the modest predictive performance of published models that use answers to household surveys as predictors. One feasible alternative is to increase the sampling period so that household features would arise as significant predictors of long-term airborne endotoxin levels.

  2. Exposure to Dust and Endotoxin of Employees in Cucumber and Tomato Nurseries

    PubMed Central

    Madsen, A. M.; Hansen, V. M.; Nielsen, S. H.; Olsen, T. T.

    2009-01-01

    Exposure to bioaerosols in occupational settings is associated with a range of adverse health effects. The aim of this study was to investigate the exposure levels to dust and endotoxin of people working in two cucumber nurseries and two tomato nurseries. Exposure was measured for greenhouse workers (n = 70) mainly working on harvesting cucumbers and tomatoes and clearing the plants after the harvest season. The people were exposed to between 0.2 and 15 mg inhalable dust m−3 (median = 1.6 mg m−3) and between 0.5 and 400 ng inhalable endotoxin m−3 (median = 32 ng m−3). The exposure to ‘total dust’ and endotoxin measured by stationary samplers (n = 30) in the greenhouses was low. Endotoxin was present in relatively high concentrations on cucumber leaves compared with leaves on pot plants. The Danish occupational exposure limit (OEL) for total organic dust is 3 mg m−3 and 36% and 17% of the cucumber and tomato workers, respectively, were exposed to >3.0 mg inhalable dust m−3. There is no OEL for endotoxin, but ‘no effect levels’ at ∼15 ng m−3 have been found. The majority of subjects (65%) were exposed to >15 ng m−3. Significantly higher exposure was found for employees in cucumber nurseries than for employees in tomato nurseries. Clearing tomato plants after the harvest season caused a higher exposure to endotoxin than tomato harvesting. In conclusion, people working in cucumber and tomato nurseries were often exposed to high levels of inhalable dust and endotoxin. Cucumber harvest workers were exposed to significantly more dust and endotoxin than tomato harvest workers. The dust and endotoxin aerosolized during the working processes were only transported to other areas in the greenhouses to a very low degree. Cucumber and tomato leaves were identified as endotoxin reservoirs. PMID:19033558

  3. Budesonide inhalation ameliorates endotoxin-induced lung injury in rabbits

    PubMed Central

    Gao, Wei

    2015-01-01

    Acute respiratory distress syndrome (ARDS) is a serious clinical problem that has a 30–50% mortality rate. Budesonide has been used to reduce lung injury. This study aims to investigate the effects of nebulized budesonide on endotoxin-induced ARDS in a rabbit model. Twenty-four rabbits were randomized into three groups. Rabbits in the control and budesonide groups were injected with endotoxin. Thereafter, budesonide or saline was instilled, ventilated for four hours, and recovered spontaneous respiratory. Peak pressure, compliance, and PaO2/FiO2 were monitored for 4 h. After seven days, PaO2/FiO2 ratios were measured. Wet-to-dry weight ratios, total protein, neutrophil elastase, white blood cells, and percentage of neutrophils in BALF were evaluated. TNF-α, IL-1β, IL-8, and IL-10 in BALF were detected. Lung histopathologic injury and seven-day survival rate of the three groups were recorded. Peak pressure was downregulated, but compliance and PaO2/FiO2 were upregulated by budesonide. PaO2/FiO2 ratios significantly increased due to budesonide. Wet-to-dry weight ratios, total protein, neutrophil elastase, white blood cells and percentage of neutrophils in BALF decreased in the budesonide group. TNF-α, IL-1β, and IL-8 levels decreased in BALF, while IL-10 levels increased in the budesonide group. Lung injuries were reduced and survival rate was upregulated by budesonide. Budesonide effectively ameliorated respiratory function, attenuated endotoxin-induced lung injury, and improved the seven-day survival rate. PMID:25956681

  4. Detection of endotoxin using a photonic crystal nanolaser

    NASA Astrophysics Data System (ADS)

    Takahashi, Daichi; Hachuda, Shoji; Watanabe, Takumi; Nishijima, Yoshiaki; Baba, Toshihiko

    2015-03-01

    Fast and reliable detection of endotoxin (ET) in medical equipment and pharmaceutical products is an essential precursor to clinical treatment. In this study, we demonstrate the use of shifts in wavelength of photonic crystal nanolasers for sensing the Limulus amebocyte lysate reaction, which is a standard method for detecting ET. From working curves of wavelength shift vs ET concentration, whose correlation factors were as high as 98%, we detected a required concentration of 0.001 EU/ml within 33 min and detected a low concentration of 0.0001 EU/ml.

  5. Biological and Chemical Characterization of Endotoxin from Capnocytophaga sputigena

    PubMed Central

    Stevens, R. H.; Sela, M. N.; McArthur, W. P.; Nowotny, A.; Hammond, B. F.

    1980-01-01

    An endotoxin was isolated from Capnocytophaga sputigena strain 4 by a modification of the hot phenol-water method. The extraction procedure yielded a lipopolysaccharide which accounted for approximately 1.5% of the dry weight of the cells. The material was composed of 18.6% lipid (as C15 fatty acid), 46.5% neutral sugar including 9.6% hexose, 18.3% 6-deoxy sugar, 1.0% 2-keto-3-deoxy sugar, and 4.8% heptose. Hexosamine, protein, and phosphorus were found in quantities amounting to 9.0, 2.9, and 2.0% of the dry weight, respectively. No pentose or nucleic acid was detected. Acid hydrolysis resulted in the release of the constituent sugars and the formation of an insoluble precipitate. The lipopolysaccharide was tested for numerous biological activities characteristic of endotoxins. The pyrogenicity was relatively low; the fever index 40 was 17 μg, and 10 μg was required to give the characteristic biphasic fever response. The toxicity of the extract was very low, with a 50% chicken embryo lethal dose of 15.6 μg and a 50% mouse embryo lethal dose of greater than 8 mg. Similarly, the C. sputigena endotoxin had modest effects on leukocytes when compared with endotoxin standards from other organisms. The extract exhibited little or no mitogenicity when tested on mouse spleen lymphocytes. It was not toxic to human peripheral polymorphonuclear leukocytes and caused the release of only a small (13%) portion of lysosomal enzymes. Although the C. sputigena lipopolysaccharide caused significant activation of mouse peritoneal macrophages, the dose required was twice that of an Escherichia coli endotoxic standard. However, the Limulus amoebocyte lysate clotting activity of the lipopolysaccharide was comparable to that of an Serratia marcescens lipopolysaccharide standard, and passive hemagglutination tests revealed that 1 μg of the lipopolysaccharide was capable of sensitizing 1 ml of a 2% sheep erythrocyte suspension for agglutination with an antiserum prepared against C

  6. Detection of endotoxin using a photonic crystal nanolaser

    SciTech Connect

    Takahashi, Daichi; Hachuda, Shoji; Watanabe, Takumi; Nishijima, Yoshiaki; Baba, Toshihiko

    2015-03-30

    Fast and reliable detection of endotoxin (ET) in medical equipment and pharmaceutical products is an essential precursor to clinical treatment. In this study, we demonstrate the use of shifts in wavelength of photonic crystal nanolasers for sensing the Limulus amebocyte lysate reaction, which is a standard method for detecting ET. From working curves of wavelength shift vs ET concentration, whose correlation factors were as high as 98%, we detected a required concentration of 0.001 EU/ml within 33 min and detected a low concentration of 0.0001 EU/ml.

  7. The ability of endotoxin adsorption during a longer duration of direct hemoperfusion with a polymyxin B-immobilized fiber column in patients with septic shock.

    PubMed

    Shimizu, Tomoharu; Obata, Toru; Sonoda, Hiromichi; Akabori, Hiroya; Tabata, Takahisa; Eguchi, Yutaka; Endo, Yoshihiro; Tani, Tohru

    2013-12-01

    The patients' hemodynamic conditions of septic shock due to intra-abdominal infection were improved by the longer duration of direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX), reducing plasma endotoxins measured by the novel endotoxin detection method, named endotoxin scattering photometry (ESP) method; however, turbidimetric method could not detect endotoxins. We also observed the reduction in the endotoxin after passing through column by ESP method even after the longer duration of PMX. ESP method may more sensitively detect endotoxins than the ordinary turbidimetric method. Moreover, we demonstrated the ability of endotoxin adsorption in spite of the longer duration of PMX.

  8. Assessing Fungal Population in Soil Planted with Cry1Ac and CPTI Transgenic Cotton and Its Conventional Parental Line Using 18S and ITS rDNA Sequences over Four Seasons

    PubMed Central

    Qi, Xiemin; Liu, Biao; Song, Qinxin; Zou, Bingjie; Bu, Ying; Wu, Haiping; Ding, Li; Zhou, Guohua

    2016-01-01

    Long-term growth of genetically modified plants (GMPs) has raised concerns regarding their ecological effects. Here, FLX-pyrosequencing of region I (18S) and region II (ITS1, 5.8S, and ITS2) rDNA was used to characterize fungal communities in soil samples after 10-year monoculture of one representative transgenic cotton line (TC-10) and 15-year plantation of various transgenic cotton cultivars (TC-15mix) over four seasons. Soil fungal communities in the rhizosphere of non-transgenic control (CC) were also compared. No notable differences were observed in soil fertility variables among CC, TC-10, and TC-15mix. Within seasons, the different estimations were statistically indistinguishable. There were 411 and 2 067 fungal operational taxonomic units in the two regions, respectively. More than 75% of fungal taxa were stable in both CC and TC except for individual taxa with significantly different abundance between TC and CC. Statistical analysis revealed no significant differences between CC and TC-10, while discrimination of separating TC-15mix from CC and TC-10 with 37.86% explained variance in PCoA and a significant difference of Shannon indexes between TC-10 and TC-15mix were observed in region II. As TC-15mix planted with a mixture of transgenic cottons (Zhongmian-29, 30, and 33B) for over 5 years, different genetic modifications may introduce variations in fungal diversity. Further clarification is necessary by detecting the fungal dynamic changes in sites planted in monoculture of various transgenic cottons. Overall, we conclude that monoculture of one representative transgenic cotton cultivar may have no effect on fungal diversity compared with conventional cotton. Furthermore, the choice of amplified region and methodology has potential to affect the outcome of the comparison between GM-crop and its parental line. PMID:27462344

  9. Assessing Fungal Population in Soil Planted with Cry1Ac and CPTI Transgenic Cotton and Its Conventional Parental Line Using 18S and ITS rDNA Sequences over Four Seasons.

    PubMed

    Qi, Xiemin; Liu, Biao; Song, Qinxin; Zou, Bingjie; Bu, Ying; Wu, Haiping; Ding, Li; Zhou, Guohua

    2016-01-01

    Long-term growth of genetically modified plants (GMPs) has raised concerns regarding their ecological effects. Here, FLX-pyrosequencing of region I (18S) and region II (ITS1, 5.8S, and ITS2) rDNA was used to characterize fungal communities in soil samples after 10-year monoculture of one representative transgenic cotton line (TC-10) and 15-year plantation of various transgenic cotton cultivars (TC-15mix) over four seasons. Soil fungal communities in the rhizosphere of non-transgenic control (CC) were also compared. No notable differences were observed in soil fertility variables among CC, TC-10, and TC-15mix. Within seasons, the different estimations were statistically indistinguishable. There were 411 and 2 067 fungal operational taxonomic units in the two regions, respectively. More than 75% of fungal taxa were stable in both CC and TC except for individual taxa with significantly different abundance between TC and CC. Statistical analysis revealed no significant differences between CC and TC-10, while discrimination of separating TC-15mix from CC and TC-10 with 37.86% explained variance in PCoA and a significant difference of Shannon indexes between TC-10 and TC-15mix were observed in region II. As TC-15mix planted with a mixture of transgenic cottons (Zhongmian-29, 30, and 33B) for over 5 years, different genetic modifications may introduce variations in fungal diversity. Further clarification is necessary by detecting the fungal dynamic changes in sites planted in monoculture of various transgenic cottons. Overall, we conclude that monoculture of one representative transgenic cotton cultivar may have no effect on fungal diversity compared with conventional cotton. Furthermore, the choice of amplified region and methodology has potential to affect the outcome of the comparison between GM-crop and its parental line. PMID:27462344

  10. Turbulent heat exchanger {Delta}T and {Delta}P

    SciTech Connect

    Steinmeyer, D.

    1996-12-31

    Optimum pressure drop ({Delta}P) and temperature difference ({Delta}T) in turbulent flow heat exchangers are presented in three frameworks: as quantitatively defined by fluid properties, the value of energy and the cost of heat exchange surface (with a little help from a relationship between [power/mass] and heat transfer); as the energy cost for heat recovery (with the {Delta}T cost being about equal to the heat exchanger cost and the {Delta}P cost being about 1/3 as great); and as the second law lost work inherent in heat exchange (with the {Delta}T loss being {approximately}3 times the {Delta}T loss).

  11. Ganges River Delta

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The Ganges River forms an extensive delta where it empties into the Bay of Bengal. The delta is largely covered with a swamp forest known as the Sunderbans, which is home to the Royal Bengal Tiger. It is also home to most of Bangladesh, one of the world's most densely populated countries. Roughly 120 million people live on the Ganges Delta under threat of repeated catastrophic floods due to heavy runoff of meltwater from the Himalayas, and due to the intense rainfall during the monsoon season. This image was acquired by Landsat 7's Enhanced Thematic Mapper plus (ETM+) sensor on February 28, 2000. This is a false-color composite image made using green, infrared, and blue wavelengths. Image provided by the USGS EROS Data Center Satellite Systems Branch

  12. Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis

    PubMed Central

    Zhang, Wenbo; Baban, Babak; Rojas, Modesto; Tofigh, Sohrab; Virmani, Suvika K.; Patel, Chintan; Behzadian, M. Ali; Romero, Maritza J.; Caldwell, Robert W.; Caldwell, Ruth B.

    2009-01-01

    Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity. PMID:19590038

  13. Advances and needs for endotoxin-free production strains.

    PubMed

    Taguchi, Seiichi; Ooi, Toshihiko; Mizuno, Kouhei; Matsusaki, Hiromi

    2015-11-01

    The choice of an appropriate microbial host cell and suitable production conditions is crucial for the downstream processing of pharmaceutical- and food-grade products. Although Escherichia coli serves as a highly valuable leading platform for the production of value-added products, like most Gram-negative bacteria, this bacterium contains a potent immunostimulatory lipopolysaccharide (LPS), referred to as an endotoxin. In contrast, Gram-positive bacteria, notably Bacillus, lactic acid bacteria (LAB), Corynebacterium, and yeasts have been extensively used as generally recognized as safe (GRAS) endotoxin-free platforms for the production of a variety of products. This review summarizes the currently available knowledge on the utilization of these representative Gram-positive bacteria for the production of eco- and bio-friendly products, particularly natural polyesters, polyhydroxyalkanoates, bacteriocins, and membrane proteins. The successful case studies presented here serve to inspire the use of these microorganisms as a main-player or by-player depending on their individual properties for the industrial production of these desirable targets. PMID:26362682

  14. Nonabsorbable Antibiotics Reduce Bacterial and Endotoxin Translocation in Hepatectomised Rats

    PubMed Central

    Kakkos, S. K.; Kirkilesis, J.; Scopa, C. D.; Arvaniti, A.; Alexandrides, T.

    1997-01-01

    There is increasing evidence that septic complications, occurring after major hepatectomies, may be caused by gram negative bacteria, translocating from the gut. We investigated in rats, the effect of extended hepatectomy on the structure and morphology of the intestinal mucosa as well as on the translocation of intestinal bacteria and endotoxins. We also examined the effect of nonabsorbable antibiotics on reducing the intestinal flora and consequently the phenomenon of translocation by administering neomycin sulphate and cefazoline. Hepatectomy was found to increase translocation, while administration of nonabsorbable antibiotics decreased it significantly. In addition, hepatectomy increased the aerobic cecal bacterial population, which normalised in the group receiving antibiotics. Among the histological parameters evaluated, villus height demonstrated a significant reduction after hepatectomy, while the number of villi per cm and the number of mitoses per crypt, remained unchanged. Our results indicate that administration of nonabsorbable antibiotics presents a positive effect on bacterial and endotoxin translocation after extended hepatectomy, and this may be related to reduction of colonic bacterial load as an intraluminal effect of antibiotics. PMID:9298382

  15. Myocardial adrenergic responsiveness after lethal and nonlethal doses of endotoxin

    SciTech Connect

    Shepherd, R.E.; Lang, C.H.; McDonough, K.H.

    1987-02-01

    A dose-dependent impairment of intrinsic myocardial performance has been observed following in vivo administration of endotoxin. The present study reports a dose-dependent increase in plasma catecholamines following endotoxin (ET) that may impair ..beta..-adrenergic responsiveness. Hearts were removed from pentobarbital-anesthetized rats 4 h after a bolus injection of saline or ET and were studied as isolated cell preparations following collagenase digestion. Responsiveness of isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in myocytes prepared from hearts of animals injected with 10 and 100 ..mu..g ET was decreased when compared with control rats and was significantly blunted in myocytes prepared from animals receiving 1000 ..mu..g ET. Similar sensitivities of the cAMP system existed, as judged by similar half-maximum effective concentration values. cAMP accumulation in the presence of 1 ..mu..M forskolin was depressed in myocytes from the 1000-..mu..g ET animals; ..beta..-adrenergic receptor density was decreased 25% in myocytes from high-dose ET animals when compared with control animals. This was accompanied by a nonsignificant reduction in the affinity of binding sites for (+/-)(/sup 3/H)CGP 12177. The blunted myocyte hormonal responsiveness following ET challenge appears to be related to the decreased activity of the adenylate cyclase that may be attributed to alterations in both receptor density and in the adenylate cyclase itself.

  16. Left ventricular performance in endotoxin shock in dogs.

    PubMed

    Guntheroth, W G; Jacky, J P; Kawabori, I; Stevenson, J G; Moreno, A H

    1982-02-01

    Endotoxin shock, with maximal velocity of contraction (Vmax) as our index of contractility, showed no myocardial depression in an earlier 4-h study (Guntheroth, Proc. Soc. Exp. Biol. Med. 157: 610--614, 1978). Because of reports of late deterioration, we studied six dogs until spontaneous death (9--18 h). Heart rate nearly doubled and left ventricular filling pressure and aortic mean pressure fell, but Vmax did not change significantly. Because of concern that the marked increase in heart rate may have contributed to an artifactual maintenance of Vmax (due to its frequency dependence, inherent in dp/dt), we studied a final group of five dogs with three additional indicators of contractility. End-systolic pressure-diameter ratio (Emax), ejection fraction (sonar-determined from the minor axis of the left ventricle), and frequency-normalized average rate of generation of power density (FARPD) all fell early and remained low until death. We conclude that myocardial contractility is significantly reduced in endotoxin shock, early and sustained. Its presence is masked somewhat in the untreated subject by the reduced work load, secondary to hypovolemia.

  17. Salivary α-amylase response to endotoxin administration in humans.

    PubMed

    Grigoleit, Jan-Sebastian; Kullmann, Jennifer S; Oberbeck, Reiner; Schedlowski, Manfred; Engler, Harald

    2013-09-01

    Salivary α-amylase (sAA) is a digestive enzyme that plays also an important role in mucosal immunity. Secretion of the sAA is largely under the control of the autonomic nervous system and increases in sAA activity have repeatedly been observed in response to various stressors. The present study aimed at investigating whether and to what extent sAA activity levels are affected during systemic inflammation. Fourteen healthy male volunteers received intravenous injections of either bacterial endotoxin or placebo at two different occasions in a randomized and double-blinded manner. sAA activity was monitored over a period of 6h together with inflammatory markers, plasma norepinephrine (NE) and salivary cortisol levels, vital parameters, and state anxiety. Endotoxin administration elicited a transient inflammatory response reflected by increases in body temperature, whole blood cell counts, and circulating levels of interleukin (IL)-6. The immune changes were accompanied by a transient increase in sAA activity, elevations in salivary cortisol and plasma NE concentrations, as well as increases in heart rate and state anxiety. Although sAA and plasma NE responses showed distinct time courses, a significant positive correlation over the total observation period was found. Whether the observed sAA response is driven by an increase in sympathetic activity or more generally reflects inflammation induced changes in sympathetic-parasympathetic balance remains to be elucidated.

  18. Endotoxin-induced endothelial fibrosis is dependent on expression of transforming growth factors β1 and β2.

    PubMed

    Echeverría, César; Montorfano, Ignacio; Tapia, Pablo; Riedel, Claudia; Cabello-Verrugio, Claudio; Simon, Felipe

    2014-09-01

    During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases.

  19. Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

    PubMed Central

    Echeverría, César; Montorfano, Ignacio; Tapia, Pablo; Riedel, Claudia; Cabello-Verrugio, Claudio

    2014-01-01

    During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases. PMID:24935972

  20. Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin.

    PubMed

    Clark, T M; Hutchinson, M J; Huegel, K L; Moffett, S B; Moffett, D F

    2005-12-01

    Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus ("stomach") region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 microm in diameter (mean 3.3+/-0.53 microm, N=12), blisters 16.9+/-1.54 microm in diameter (N=10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of delta-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.

  1. Effects of endotoxin on mammary secretion of lactating cows. [Escherichia coli

    SciTech Connect

    Lengemann, F.W.; Pitzrick, M.

    1986-05-01

    The objectives were to describe the magnitude and time course of changes in milk pH, Na, K, lactose, and somatic cells and to determine if paracellular pathways were altered after infusion of Escherichia coli endotoxin (serotype 0128:AB12) to produce inflammation in one-half of the udder of the goat. Intramammary infusion of endotoxin increased pH, number of somatic cells, and Na and decreased K and lactose in milk. Sodium and number of somatic cells were increased by as little as .1..mu..g of endotoxin; .25 ..mu..g produced changes in most of the other parameters; maximal effect was elicited by 1..mu..g of endotoxin. The gland response peaked from 5 to 7 h after infusion of endotoxin with an increase in milk cellularity as the only significant effect noted in the control gland. Infusion of (/sup 14/C)lactose into the gland and (/sup 99m/Tc)albumin into the blood demonstrated that large molecules were more able to cross into and out of udder halves after endotoxin treatment. It is suggested that ion interchange rather than bulk flow across paracellular paths is responsible for changes. In addition, endotoxin appeared to reduce lactose secretion and synthesis.

  2. In silico designed nanoMIP based optical sensor for endotoxins monitoring.

    PubMed

    Abdin, M J; Altintas, Z; Tothill, I E

    2015-05-15

    Molecular modelling was used to select specific monomers suitable for the design of molecularly imprinted polymers (MIPs) with high affinity towards endotoxins. MIPs were synthesised using solid-phase photopolymerisation with endotoxins from Escherichia coli 0111:B4 as the template. This technique also allowed the endotoxin template to be reused successfully. Particle size of ~190-220 nm was achieved with low polydispersity index, which confirms the quality of the produced MIPs. For the development of the optical sensor, SPR-2 biosensor system was used by functionalising the gold sensor chip with the MIP nanoparticles using EDC/NHS coupling procedure. The affinity based-endotoxin assay can detect endotoxins in the concentration range of 15.6-500 ng mL(-1). MIP surfaces were regenerated showing stability of the method for subsequent analysis and dissociation constants were calculated as 3.24-5.24×10(-8) M. The developed SPR sensor with the novel endotoxins nanoMIP showed the potential of the technology for endotoxins capture, detection and risk management and also the importance of computational modelling to design the artificial affinity ligands. PMID:25155060

  3. Measurement of endotoxins in bioaerosols at workplace: a critical review of literature and a standardization issue.

    PubMed

    Duquenne, Philippe; Marchand, Geneviève; Duchaine, Caroline

    2013-03-01

    Endotoxins are lipopolysaccharides found in the outer membrane of most Gram-negative bacteria and cyanobacteria. Worker exposure to endotoxins has been shown in a number of work situations and is associated with both respiratory and systemic pathologies. The lack of an occupational exposure limit is mainly due to the absence of a standard protocol at the international level for sampling and analyzing airborne endotoxins. The bibliographic review in this article takes an exhaustive look at the current knowledge on measuring airborne endotoxins. It shows that, despite several reference documents at the international level, the methods used to measure endotoxin exposure differ considerably from one laboratory to another. Standardization is necessary to reduce interlaboratory variability and, ultimately, to improve the use of interstudy data. The bibliographic review presents the current status of standardization for airborne endotoxin measurement methods in the workplace and summarizes areas for further research. This article is both a reference document for all operators wishing to use such methods and a working document to build international consensus around the measurement of airborne endotoxins.

  4. In silico designed nanoMIP based optical sensor for endotoxins monitoring.

    PubMed

    Abdin, M J; Altintas, Z; Tothill, I E

    2015-05-15

    Molecular modelling was used to select specific monomers suitable for the design of molecularly imprinted polymers (MIPs) with high affinity towards endotoxins. MIPs were synthesised using solid-phase photopolymerisation with endotoxins from Escherichia coli 0111:B4 as the template. This technique also allowed the endotoxin template to be reused successfully. Particle size of ~190-220 nm was achieved with low polydispersity index, which confirms the quality of the produced MIPs. For the development of the optical sensor, SPR-2 biosensor system was used by functionalising the gold sensor chip with the MIP nanoparticles using EDC/NHS coupling procedure. The affinity based-endotoxin assay can detect endotoxins in the concentration range of 15.6-500 ng mL(-1). MIP surfaces were regenerated showing stability of the method for subsequent analysis and dissociation constants were calculated as 3.24-5.24×10(-8) M. The developed SPR sensor with the novel endotoxins nanoMIP showed the potential of the technology for endotoxins capture, detection and risk management and also the importance of computational modelling to design the artificial affinity ligands.

  5. Selective detection of endotoxin using an impedance aptasensor with electrochemically deposited gold nanoparticles.

    PubMed

    Su, Wenqiong; Kim, Sung-Eun; Cho, MiSuk; Nam, Jae-Do; Choe, Woo-Seok; Lee, Youngkwan

    2013-01-01

    Using a single-stranded DNA (ssDNA) aptamer exhibiting high binding affinity (Kd = 12 nM) to endotoxin as a probe, an impedance sensor where aptamer-conjugated gold nanoparticles (AuNPs) were electrochemically deposited on a gold electrode was fabricated and its performance in regard to endotoxin detection assessed. AuNPs have been employed widely as biosensors because of their unique physical and chemical properties. In order to maximize the performance of the impedance aptasensor on endotoxin detection, some critical factors affecting aptamer conjugation to AuNPs and target recognition ability (i.e. concentrations of aptamer coupled with AuNPs, pH, ion strength and cation effect at the time of aptamer-endotoxin interaction) were optimized. Electrochemical impendence spectroscopy, cyclic voltametry, atomic force microscope, scanning electron microscope and quartz crystal microbalance were employed to characterize all the modification/detection procedures during the sensor fabrication. The developed aptasensor showed a broad linear dynamic detection range (0.01-10.24 ng/ml) with a very low detection limit for endotoxin (0.005 ng/ml), despite the presence of several biomolecules (e.g. plasmid DNA, RNA, serum albumin, Glc and sucrose) known to interfere with other endotoxin assays. The demonstrated aptasensor required a detection time of only 10 min, providing a simple and fast analytical method to specifically detect endotoxin from complex biological liqors.

  6. Quantification of the Influence of Endotoxins on the Mechanics of Adult and Neonatal Red Blood Cells.

    PubMed

    Ito, Hiroaki; Kuss, Navina; Rapp, Bastian E; Ichikawa, Masatoshi; Gutsmann, Thomas; Brandenburg, Klaus; Pöschl, Johannes M B; Tanaka, Motomu

    2015-06-25

    In this study, we physically modeled the influence of endotoxin-induced sepsis symptoms on human red blood cells (RBCs) by quantifying the impact of endotoxins on the cell mechanics by the analysis of Fourier-transformed mean square amplitude of shape fluctuation, called flicker spectroscopy. With the aid of a microfluidic diffusion chamber, we noninvasively determined principal mechanical parameters of human RBCs in the absence and presence of endotoxins for individual RBCs for the first time. Because of the elongation of saccharide chain length of endotoxins, we found an increase in the morphological transition from discocytes to echinocytes, and monotonic changes in the mechanical parameters. Since septic shocks often cause lethal risks of neonates, we measured the mechanical parameters of neonatal RBCs, and compared them to those of adult RBCs. The quantitative comparison reveals that neonatal RBCs are more susceptible to the effect of endotoxins than adult RBCs. Furthermore, coincubation with the antiseptic peptide P19-2.5 (Aspidasept) with endotoxin results in a slight suppression of the impact of the endotoxin. The strategy proposed in our study can potentially be applied for the quantitative diagnosis of RBCs based on mechanical readouts.

  7. Endotoxin regulates the maturation of sterol regulatory element binding protein-1 through the induction of cytokines.

    PubMed

    Diomede, L; Albani, D; Bianchi, M; Salmona, M

    2001-01-01

    Endotoxin (LPS), by raising the levels of cytokines, markedly influences lipid metabolism. To clarify the molecular mechanism of this effect, we examined the action of endotoxin in vitro and in vivo on the regulation of sterol regulatory element binding protein-1 (SREBP-1). In HepG2 cells stimulated with LPS, a dose-dependent increase in the level of the mature form of SREBP-1 was observed. For in vivo studies, endotoxin was administered intraperitoneally to CD1 mice fed with a standard or a cholesterol-enriched diet to increase the basal levels of circulating and liver cholesterol. Endotoxin raised cholesterol levels and stimulated the maturation of hepatic SREBP-1 in both normal and cholesterol-fed mice, indicating that the lipogenic effect of LPS was independent of endogenous sterol levels. To assess whether the lipogenic effect of endotoxin was linked to cytokine production, we administered LPS to C57Bl/6J endotoxin-sensitive and to C3H/HeJ endotoxin-resistant mice, which do not produce tumor necrosis factor in response to LPS. Significant induction of cholesterol levels and SREBP-1 activation was observed only in C57Bl/6J mice, indicating that cytokine production is crucial for the regulation of SREBP-1, and that the transcriptional activation of cholesterol biosynthesis may be part of the acute-phase response.

  8. Glibenclamide attenuates the antiarrhythmic effect of endotoxin with a mechanism not involving K(ATP) channels.

    PubMed

    Iskit, Alper B; Erkent, Ulkem; Ertunc, Mert; Guc, M Oguz; Ilhan, Mustafa; Onur, Rustu

    2007-02-01

    The role of K(ATP) channels in the antiarrhythmic effect of Escherichia coli endotoxin-induced nitric oxide synthase (iNOS) was examined in an anesthetised rat model of myocardial ischemia and reperfusion arrhythmia by using glibenclamide (1 mg kg(-1)), nateglinide (10 mg kg(-1)) and repaglinide (0.5 mg kg(-1)). Endotoxin (1 mg kg(-1)) was administered intraperitoneally 4 h before the occlusion of the left coronary artery and glibenclamide, nateglinide or repaglinide was administered 30 min before coronary artery occlusion. We also evaluated the effects of K(ATP) channel blockers and nonselective K(+) channel blocker tetraethylammonium (TEA) on cardiac action potential configuration in the atria obtained from endotoxemic rats. The mean arterial blood pressure of rats receiving endotoxin was lower during both the occlusion and reperfusion periods. Endotoxin significantly reduced the total number of ectopic beats and the duration of ventricular tachycardia. Glibenclamide, but not nateglinide and repaglinide, prevented the hypotension and antiarrhythmic effects of endotoxin. Atria obtained from endotoxin-treated rats had prolonged action potential duration. This effect was abolished with pretreatment of iNOS inhibitors, l-canavanine and dexamethasone and perfusion of glibenclamide, but not with TEA and non-sulfonylurea drug, nateglinide. We demonstrated that glibenclamide inhibits the antiarrhythmic effect of endotoxin and this effect does not appear to involve K(ATP) channels.

  9. [Bile acids and endotoxins: physico-chemical defense of the body].

    PubMed

    Bertók, L

    1999-01-01

    The toxic effects of endotoxin--the cell wall component of Gram negative intestinal bacteria--under experimental conditions can be induced only when they are administered parenterally. However, in naturally occurring enteroendotoxemic diseases (e.g. septic and various shocks, etc.), the endotoxin absorbs from the intestinal tract. The cause and mode of translocation was unknown. The generally used experimental shock models differ from natural diseases only in the mode by which endotoxin enters the blood circulation. If the common bile ducts of rats were chronically cannulated (bile deprived animals) perorally administered endotoxin was absorbed from the intestinal canal into blood circulation and provoked endotoxin shock. The translocation of endotoxins and consequent shock can be prevented by sodium deoxycholate or natural biles. The bile acids can split the endotoxin macromolecule (atoxic fragments). A similar destructive detergent action might will be a significant factor against potential infectious agents with lipoprotein outer structure (e.g. so-called "big" viruses). This defense mechanism of macrooganisms based on the detergent activity of bile acids is called as physico-chemical defense system. On the basis of this knowledge the bile acids might be used in the prevention and therapy of some clinical processes (e.g. hepatorenal syndrome; psoriasis).

  10. Characterization of the DialGuard device for endotoxin removal in hemodialysis.

    PubMed

    Szathmary, Susan; Hegyi, Edit; Amoureux, Marie-Claude; Rajapakse, Nandani; Chicorka, Lisa; Szalai, Gyorgy; Reszegi, Katalin; Derbyshire, Zachary; Paluh, Janet; Dodson, Bryce; Grandics, Peter

    2004-01-01

    Bacterial pyrogens, capable of penetrating dialyzer membranes, are responsible for a systemic inflammatory reaction in hemodialysis patients. Dialyzer reuse, involving rinsing of the dialyzer with pyrogen-containing water, may exacerbate this situation. Studies of the mechanism of action of endotoxin suggest that it irreversibly damages the vascular endothelium. The novel endotoxin removal method described here, is based on affinity-binding of endotoxin by the adsorbent ClarEtox, a USP Class VI-certified resin that is the active component of the medical device DialGuard. Under standard hemodialysis operating conditions, challenge of DialGuard with Pseudomonas maltophilia supernatant-spiked dialysate, containing 35-193 EU/ml endotoxin, resulted in endotoxin levels below 0.05 EU/ml in the treated dialysate. DialGuard was able to decrease endotoxin concentrations in the dialysate from a range of 2.39-8.49 to <0.005 EU/ml. DialGuard supports high fluid velocities at low back pressures and can be sanitized using the heat sanitization cycle of hemodialysis machines. DialGuard offers a simple, user-friendly way to reduce the concentration of endotoxin in dialysate and water for dialysis at a low cost.

  11. Selective detection of endotoxin using an impedance aptasensor with electrochemically deposited gold nanoparticles.

    PubMed

    Su, Wenqiong; Kim, Sung-Eun; Cho, MiSuk; Nam, Jae-Do; Choe, Woo-Seok; Lee, Youngkwan

    2013-01-01

    Using a single-stranded DNA (ssDNA) aptamer exhibiting high binding affinity (Kd = 12 nM) to endotoxin as a probe, an impedance sensor where aptamer-conjugated gold nanoparticles (AuNPs) were electrochemically deposited on a gold electrode was fabricated and its performance in regard to endotoxin detection assessed. AuNPs have been employed widely as biosensors because of their unique physical and chemical properties. In order to maximize the performance of the impedance aptasensor on endotoxin detection, some critical factors affecting aptamer conjugation to AuNPs and target recognition ability (i.e. concentrations of aptamer coupled with AuNPs, pH, ion strength and cation effect at the time of aptamer-endotoxin interaction) were optimized. Electrochemical impendence spectroscopy, cyclic voltametry, atomic force microscope, scanning electron microscope and quartz crystal microbalance were employed to characterize all the modification/detection procedures during the sensor fabrication. The developed aptasensor showed a broad linear dynamic detection range (0.01-10.24 ng/ml) with a very low detection limit for endotoxin (0.005 ng/ml), despite the presence of several biomolecules (e.g. plasmid DNA, RNA, serum albumin, Glc and sucrose) known to interfere with other endotoxin assays. The demonstrated aptasensor required a detection time of only 10 min, providing a simple and fast analytical method to specifically detect endotoxin from complex biological liqors. PMID:23165992

  12. Rapid diagnosis of gram negative pneumonia by assay of endotoxin in bronchoalveolar lavage fluid.

    PubMed Central

    Pugin, J; Auckenthaler, R; Delaspre, O; van Gessel, E; Suter, P M

    1992-01-01

    BACKGROUND: Diagnosis of ventilator associated pneumonia can be made by quantitative cultures of bronchoalveolar lavage fluid or of protected specimen brushings, though cultures require 24-48 hours to provide results. In 80% of cases aerobic Gram negative bacteria are the cause. METHODS: A rapid diagnostic method of assessing the endotoxin content of lavage fluid by Limulus assay is described. Forty samples of lavage fluid were obtained from patients with multiple trauma requiring mechanical ventilation for a prolonged period. Pneumonia was diagnosed on the basis of clinical, radiological, and bacteriological findings, including quantitative cultures of lavage fluid. RESULTS: A relation was observed between the concentration of endotoxin in lavage fluid and the quantity of Gram negative bacteria. The median endotoxin content of lavage fluid in Gram negative bacterial pneumonia was 15 endotoxin units (EU)/ml; the range observed in individual patients was 6 to > 150 EU/ml. In patients with pneumonia due to Gram positive cocci and in non-infected patients the median endotoxin level was 0.17 (range < or = 0.06 to 2) EU/ml. An endotoxin level greater than or equal to 6 EU/ml distinguished patients with Gram negative bacterial pneumonia from colonised patients and from those with pneumonia due to Gram positive cocci. CONCLUSION: The measurement of endotoxin in lavage fluid is a rapid (less than two hours) and accurate diagnostic method. It should allow specific and early treatment of Gram negative bacterial pneumonia. PMID:1412100

  13. Delta II Mars Pathfinder

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Final preparations for lift off of the DELTA II Mars Pathfinder Rocket are shown. Activities include loading the liquid oxygen, completing the construction of the Rover, and placing the Rover into the Lander. After the countdown, important visual events include the launch of the Delta Rocket, burnout and separation of the three Solid Rocket Boosters, and the main engine cutoff. The cutoff of the main engine marks the beginning of the second stage engine. After the completion of the second stage, the third stage engine ignites and then cuts off. Once the third stage engine cuts off spacecraft separation occurs.

  14. Removing endotoxin from plasmid samples by Triton X-114 isothermal extraction.

    PubMed

    Ma, Rui; Zhao, Jun; Du, Hui-Cong; Tian, Shuang; Li, Li-Wen

    2012-05-15

    Triton X-114 temperature transition extraction has been considered to be a simple and cost-effective strategy to eliminate endotoxin from plasmid preparations. However, a repeated cooling-heating process may promote the degradation of plasmid DNA. Based on the finding that the cloud point of Triton X-114 solution increases substantially in the presence of small amounts of sodium dodecyl sulfate (SDS) and that electrolytes decrease the cloud point of Triton X-114-SDS solution drastically, we designed a Triton X-114 isothermal extraction method for removing endotoxin from plasmid samples and found that it has the same endotoxin removal efficiency when compared with the temperature transition extraction method.

  15. The molecular adsorption-type endotoxin detection system using immobilized ɛ-polylysine

    NASA Astrophysics Data System (ADS)

    Ooe, Katsutoshi; Tsuji, Akihito; Nishishita, Naoki; Hirano, Yoshiaki

    2007-12-01

    Hemodialysis for chronic renal failure is the most popular treatment method with artificial organs. However, hemodialysis patients must continue the treatment throughout their life, the results of long term extracorporeal dialysis, those patients develop the various complications and diseases, for example, dialysis amyloidosis etc. Dialysis amyloidosis is one of the refractory complications, and endotoxin is thought to be the most likely cause of it, recently. Endotoxin is one of the major cell wall components of gram-negative bacteria, and it has various biological activities. In addition, it is known that a mount of endotoxin exists in living environment, and medicine is often contaminated with endotoxin. When contaminated dialyzing fluids are used to hemodialysis, above-mentioned dialysis amyloidosis is developed. Therefore, it is important that the detection and removal of endotoxin from dialyzing fluids. Until now, the measurement methods using Limulus Amebosyte Lysate (LAL) reagent were carried out as the tests for the presence of endtoxin. However, these methods include several different varieties of measurement techniques. The following are examples of them, gelatinization method, turbidimetric assay method, colorimetric assay method and fluoroscopic method. However, these techniques needed 30-60 minutes for the measurement. From these facts, they are not able to use as a "real-time endotoxin detector". The detection of endotoxin has needed to carry out immediately, for that reason, a new detection method is desired. In this research, we focused attention to adsorption reaction between ɛ-polylysine and endotoxin. ɛ-polylysine has the structure of straight chain molecule composed by 25-30 residues made by lysine, and it is used as an antimicrobial agent, moreover, cellulose beads with immobilized ɛ-polylysine is used as the barrier filter for endotoxin removal. The endotoxin is adsorbed to immobilized ɛ-polylysine, as the result of this reaction, the

  16. Lack of spatial variation of endotoxin in ambient particulate matter across a German metropolitan area

    NASA Astrophysics Data System (ADS)

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

    In this study, we describe the spatial variation of endotoxin across an urban setting using Geographic Information Systems (GIS) methods. We also identify potential sources of endotoxin that account for between-site variability and compare endotoxin levels in particulate matter with a 50% aerodynamic cut-off diameter of 2.5 μm (PM 2.5) and of 10 μm (PM 10). In 1999-2000, we collected PM 2.5 and PM 10 in Munich urban air and measured soluble endotoxin concentrations in both particle fractions. Using Teflon filters and Harvard impactors, PM 2.5 was collected at 40 outdoor monitoring sites across Munich and PM 10 at a subset of these sites ( n=12). Approximately four samples were collected at each site for a total of 158 PM 2.5 samples and 48 PM 10 samples. We visited and characterized the surrounding 100 m of each site for potential endotoxin sources. The geometric mean endotoxin concentration for all sites was 1.46 EU mg -1 PM 2.5 (95% confidence intervals (CI): 1.21-1.77) and at the subset of the sites was 1.30 EU mg -1 PM 2.5 (95% CI: 1.01-1.67 EU mg -1 PM 2.5). Endotoxin levels in PM 10 were higher, 3.91 EU mg -1 PM 10 (95% CI: 3.03-5.03 EU mg -1 PM 10), than in PM 2.5 and were moderately correlated, r=0.51. All endotoxin concentrations measured in this study were <5.5 EU m -3 and thus lower than the accepted thresholds for acute adverse health effects for occupational exposures. Sites with more potential sources ( n⩾3) had slightly higher mean endotoxin levels (MR: 1.30 for EU mg -1 PM 2.5 and 1.13 for EU m -3 PM 2.5) than sites with no identified sources. Based on the ranges of endotoxin levels at the different sites, we found very little spatial variation in ambient endotoxin concentrations across the metropolitan area of Munich using inverse distance weighting method (IDW) methods ( R2=0.013 for EU mg -1 PM 2.5 and R2=0.020 for EU m -3 PM 2.5). Potential sources of endotoxin surrounding the sites only partly explained the variation seen.

  17. Deleterious effects of endotoxin on cultured endothelial cells: an in vitro model of vascular injury

    SciTech Connect

    Yamada, O.; Moldow, C.F.; Sacks, T.; Craddock, P.R.; Boogaerts, M.A.; Jacob, H.S.

    1981-06-01

    The effects of endotoxin-triggered granulocytes on the viability of endothelial cells in vitro was investigated. Endotoxin or its lipid A component caused granulocytes to adhere to and significantly damage cultured endothelial cells. Fresh serum is not necessary but does amplify both adherence and endothelial injury. Much of the endothelial injury was inhibited by free-radical scavengers or by blocking granulocyte adhesion to endothelial cells and appears to result from free radical production by the stimulated granulocyte. Studies in this model suggest a pathogenic role for the endotoxin-triggered granulocyte in the Shwartzman reaction and perhaps related clinical disorders.

  18. Mild alkaline hydrolysis of lipopolysaccharide endotoxin enhances its mitogencity for murine B cells.

    PubMed Central

    Goodman, G W; Sultzer, B M

    1977-01-01

    Mild alkaline hydrolysis was found to enhance the mitogenicity of lipopolysaccharide endotoxin for murine B lymphocytes. Alkaline treated lipopolysaccharide also retained its property as a polyclonal activator. Whereas this treatment reduced the lethality of endotoxin for mice, its toxicity for lymphocytes cultured in the absence of fetal calf serum was increased. Lipid analysis indicated that there were no significant changes in the fatty acids of lipid A, but particle size was significantly reduced and the material was more homogeneous and soluble than untreated lipopolysaccharide. The relationship of these effect on the structure of lipopolysaccharide endotoxin to the mechanism of B-lymphocyte activation is discussed. Images PMID:18405

  19. DELTA PHASE PLUTONIUM ALLOYS

    DOEpatents

    Cramer, E.M.; Ellinger, F.H.; Land. C.C.

    1960-03-22

    Delta-phase plutonium alloys were developed suitable for use as reactor fuels. The alloys consist of from 1 to 4 at.% zinc and the balance plutonium. The alloys have good neutronic, corrosion, and fabrication characteristics snd possess good dimensional characteristics throughout an operating temperature range from 300 to 490 deg C.

  20. IMPACT OF OBESITY ON ENDOTOXIN-INDUCED DISSEMINATED INTRAVASCULAR COAGULATION.

    PubMed

    Duburcq, Thibault; Tournoys, Antoine; Gnemmi, Viviane; Hubert, Thomas; Gmyr, Valery; Pattou, François; Jourdain, Mercé

    2015-10-01

    An early activation of coagulation and fibrinolysis occurs during sepsis, leading to the syndrome of disseminated intravascular coagulation (DIC). Obesity has been demonstrated to be a hypercoagulable and hypofibrinolytic state, but its impact on DIC has never been studied. In this study, we aimed to determine if obesity impairs DIC in an acute endotoxic shock model using minipigs. This was a prospective, comparative, and experimental ancillary study approved by the Animal Ethics Committee. Pigs were chosen as a clinically relevant species, resembling humans in coagulation reactions. Four groups of five "Yucatan" minipigs were studied: lean and obese control groups, a lean lipopolysaccharide (LPS) group receiving Escherichia coli endotoxin (LPS), and an obese LPS group receiving the same endotoxin dose. We measured standard coagulation parameters (prothrombin time [PT], platelet count, and fibrinogen levels), thrombin-antithrombin complexes, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. All measurements were performed at baseline and 30, 60, 90, 150, and 300 min. Results were given as median with interquartile ranges. At baseline, platelet count (477 [428 - 532] G/L vs. 381 [307 - 442] G/L; P = 0.005) and fibrinogen levels (4.6 [3.8 - 5.2] g/L vs. 2 [1.8 - 2.9] g/L; P < 0.001) were significantly higher, whereas PT (80% [76% - 92%] vs. 96% [89% - 100%]; P = 0.01) was significantly lower in obese pigs compared with lean pigs. In the LPS groups, administration of endotoxin resulted in a typical hypokinetic shock with DIC. The decrease in coagulation parameters (PT, platelet count, and fibrinogen levels) and the increase in thrombin-antithrombin complexes (581 [382 - 1,057] μg/mL vs. 247 [125 - 369] μg/mL at 150 min; P = 0.03) were significantly more important in the obese LPS group compared with those in the lean LPS group. Concerning the fibrinolytic reaction, we found a slightly more elevated increase of plasminogen

  1. The Devil's in the Delta

    ERIC Educational Resources Information Center

    Luyben, William L.

    2007-01-01

    Students frequently confuse and incorrectly apply the several "deltas" that are used in chemical engineering. The deltas come in three different flavors: "out minus in", "big minus little" and "now versus then." The first applies to a change in a stream property as the stream flows through a process. For example, the "[delta]H" in an energy…

  2. Delta-ALA urine test

    MedlinePlus

    Delta-aminolevulinic acid ... This test looks for an increased level of delta-ALA. It may be used to help diagnose ... An increased level of urinary delta-ALA may indicate: Lead poisoning ... level may occur with chronic (long-term) liver disease .

  3. Collaborative study for the establishment of the WHO 3(rd) International Standard for Endotoxin, the Ph. Eur. endotoxin biological reference preparation batch 5 and the USP Reference Standard for Endotoxin Lot H0K354.

    PubMed

    Findlay, L; Desai, T; Heath, A; Poole, S; Crivellone, M; Hauck, W; Ambrose, M; Morris, T; Daas, A; Rautmann, G; Buchheit, K H; Spieser, J M; Terao, E

    2015-01-01

    An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and

  4. Novel endotoxin assay by laser light-scattering particle-counting method.

    PubMed

    Mitsumoto, Kotaro; Yabusaki, Katsumi; Kobayashi, Koji; Shirasawa, Yoshiaki; Obata, Toru

    2009-01-01

    Exposure of Limulus amoebocyte lysate to endotoxin under stirring produced light-reflective particles that appeared to be coagulin polymers. A laser light-scattering particle counter, the PA-200, detected these particles sensitively. The PA-200 detected endotoxin at a concentration as low as 0.00015 EU/ml in 71 min, whereas the minimum endotoxin concentration measured by a turbidimeter, ET-2000, was 0.0005 EU/ml in 138 min. Moreover, PA-200 was much less affected by the presence of colored substances and refractive materials than was ET-2000. We propose that the high sensitivity, speed, and high interference tolerance of the laser light-scattering particle-counting method make it more useful than the widely used turbidimetric method for quantitative endotoxin assay.

  5. Augmentation of endotoxin fever by recombinant human beta interferon in rabbits.

    PubMed Central

    Kawasaki, H; Moriyama, M; Tanaka, A

    1987-01-01

    Nonpyrogenic amounts of endotoxin (0.1 to 1 ng/kg), hardly detectable by conventional Limulus amoebocyte lysate tests, could produce a fever of around 1 degree C when injected with a nonpyrogenic dose (6 X 10(5) U/kg) of recombinant human beta interferon (IFN-beta) in rabbits. Release of endogenous IFN and tumor necrosis factor by endotoxin was also dramatically increased by recombinant human IFN-beta, and their levels in the blood were closely correlated with the increase of body temperature. These data suggest, if the synergism between IFN and endotoxin also operates in the homologous system (human IFN-human cells), that contaminating endotoxin in IFNs, even if not detectable by Limulus amoebocyte lysate test, can contribute to IFN fever to a considerable extent in humans. PMID:2437032

  6. The influence of bacterial exotoxins and endotoxins on the phagocytic activity of human macrophages in culture.

    PubMed

    D'Onofrio, C; Paradisi, F

    1983-01-01

    The effect of bacterial exotoxins and endotoxins on phagocytosis was tested on human macrophages in monolayer cultures by determining the rate of zymosan particle ingestion at different toxin concentrations and incubation times. The exotoxins tested were staphylococcal alpha-toxin and diphtheria-toxin. The endotoxins used were lipopolysaccharides from Salmonella typhi, Salmonella typhimurium, Shigella flexneri and Serratia marcescens. Phagocytosis was significantly impaired after prolonged incubation with diphtheria toxin whereas alpha-toxin was ineffective. Endotoxin-treated macrophages showed a wide range of phagocytic activity. Enhancement of phagocytosis was observed with a low concentration of endotoxin (1 microgram/ml) from S. typhi, S. typhimurium and S. flexneri. Higher concentrations (2.5 and 5 micrograms/ml) depressed phagocytosis to varying extents, except for S. typhi lipopolysaccharide, which did not induce a significant decrease in phagocytosis in comparison to the controls.

  7. Altered hepatic vasopressin and alpha 1-adrenergic receptors after chronic endotoxin infusion

    SciTech Connect

    Roth, B.L.; Spitzer, J.A.

    1987-05-01

    Sepsis and septic shock are complicated by a number of hemodynamic and metabolic aberrations. These include catecholamine refractoriness and altered glucose metabolism. Recently, a nonshock rat model of continuous endotoxin infusion via an implanted osmotic pump was developed that reproduces some of the metabolic and cardiovascular findings of human sepsis. By using this model, we have found a decreased number of hepatic plasma membrane alpha 1-adrenergic and (Arg8)vasopressin receptors in rats continuously infused with endotoxin. There was a significant decrease in (/sup 3/H)prazosin (35 +/- 7%) and (/sup 3/H) (Arg8)vasopressin (43 +/- 8%) receptors after 30 h of continuous endotoxin infusion with no change in affinity. The ability of norepinephrine to form the high-affinity complex with alpha 1-adrenergic receptors was not altered after chronic endotoxin infusion. The results are consistent with the concept that alterations in receptor number might underlie certain of the metabolic consequences of chronic sepsis.

  8. High mortality rates occur in copper deficient rats exposed to a normally nonlethal endotoxin treatment

    SciTech Connect

    DiSilvestro, R.; Joseph, E.; Yang, F.L. )

    1991-03-15

    Endotoxin hepatotoxicity is proposed to occur by processes which could be retarded by 3 copper enzymes: ceruloplasmin, Cu-Zn superoxide dismutase (SOD), and extracellular (EC) SOD. Weanling rats fed low copper for 40 days showed low activity levels of these enzymes, and a very high mortality rate 20 h after endotoxin injection. No rats fed adequate copper died from this treatment. In addition, serum transaminase activities, indicators of liver damage, were elevated by 3 h to a greater extent in the deficient rats than in the adequates. The high susceptibility to endotoxemia in the deficient rats was not associated with low hepatic glutathione, high liver malondialedhyde, nor restricted metallothionein induction 3 h after endotoxin injection. Endotoxin reduced serum EC SOD activities in adequate and deficient rats, but final values were lower in the latter. Studies on roles of specific copper enzymes in resistance to endotoxemia are currently underway.

  9. Evaluation Of Airborne Endotoxin Concentrations Associated With Management Of Crop Grown On Applied Biosolids

    EPA Science Inventory

    Public health concerns have been expressed regarding inhalation exposure associated with the application of biosolids on cropland, which is due to the potential aerosolization of microorganisms, cell wall products, volatile chemicals, and nuisance odors. Endotoxin is a component...

  10. DELTAS: A new Global Delta Sustainability Initiative (Invited)

    NASA Astrophysics Data System (ADS)

    Foufoula-Georgiou, E.

    2013-12-01

    Deltas are economic and environmental hotspots, food baskets for many nations, home to a large part of the world population, and hosts of exceptional biodiversity and rich ecosystems. Deltas, being at the land-water interface, are international, regional, and local transport hubs, thus providing the basis for intense economic activities. Yet, deltas are deteriorating at an alarming rate as 'victims' of human actions (e.g. water and sediment reduction due to upstream basin development), climatic impacts (e.g. sea level rise and flooding from rivers and intense tropical storms), and local exploration (e.g. sand or aggregates, groundwater and hydrocarbon extraction). Although many efforts exist on individual deltas around the world, a comprehensive global delta sustainability initiative that promotes awareness, science integration, data and knowledge sharing, and development of decision support tools for an effective dialogue between scientists, managers and policy makers is lacking. Recently, the international scientific community proposed to establish the International Year of Deltas (IYD) to serve as the beginning of such a Global Delta Sustainability Initiative. The IYD was proposed as a year to: (1) increase awareness and attention to the value and vulnerability of deltas worldwide; (2) promote and enhance international and regional cooperation at the scientific, policy, and stakeholder level; and (3) serve as a launching pad for a 10-year committed effort to understand deltas as complex socio-ecological systems and ensure preparedness in protecting and restoring them in a rapidly changing environment. In this talk, the vision for such an international coordinated effort on delta sustainability will be presented as developed by a large number of international experts and recently funded through the Belmont Forum International Opportunities Fund. Participating countries include: U.S., France, Germany, U.K., India, Japan, Netherlands, Norway, Brazil, Bangladesh

  11. Mononuclear cells in the corneal response to endotoxin

    SciTech Connect

    Howes, E.L.; Cruse, V.K.; Kwok, M.T.

    1982-04-01

    A severe keratitis can be produced after the direct injection of bacterial endotoxin, or lipopolysaccharide (LPS), in rabbits. Corneal inflammation can progress to scarring and vascularization within a 2 to 3 week period. Pretreatment with systemic adrenal corticosteroids (triamcinolone) prevents this response. Limbal cellular and vascular events were studied during the first 20 hr after injection of LPS in treated and nontreated rabbits. Perivascular limbal inflammatory cells were counted and limbal vascular permeability was assessed by extravasation of 131I-albumin and 125I-fibrinogen in the cornea. Corticosteroids decreased but did not prevent the early protein extravasation and profoundly altered the inflammatory cell population around blood vessels at the limbus. Mononuclear cells, particularly mononuclear phagocytes, were sharply reduced. It is proposed that these cell types play an important role in the perpetuation and amplification of the inflammatory response in this reaction.

  12. Biophysical Mechanisms of Endotoxin Neutralization by Cationic Amphiphilic Peptides

    PubMed Central

    Kaconis, Yani; Kowalski, Ina; Howe, Jörg; Brauser, Annemarie; Richter, Walter; Razquin-Olazarán, Iosu; Iñigo-Pestaña, Melania; Garidel, Patrick; Rössle, Manfred; Martinez de Tejada, Guillermo; Gutsmann, Thomas; Brandenburg, Klaus

    2011-01-01

    Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS. PMID:21641310

  13. Residential indoor and outdoor coarse particles and associated endotoxin exposures

    NASA Astrophysics Data System (ADS)

    Wheeler, Amanda J.; Dobbin, Nina A.; Lyrette, Ninon; Wallace, Lance; Foto, Mark; Mallick, Ranjeeta; Kearney, Jill; Van Ryswyk, Keith; Gilbert, Nicolas L.; Harrison, Ian; Rispler, Kathleen; Héroux, Marie-Eve

    2011-12-01

    There is a growing body of evidence demonstrating that coarse particles (PM 10-2.5) have detrimental impacts upon health, especially for respiratory effects. There are limited data available for indoor residential exposures. Some data exist regarding the composition of this PM size fraction with emphasis on crustal elements and biological components. This study includes data from 146 homes sampled in Regina, Saskatchewan (SK) where 5-day integrated concurrent monitoring of indoor and outdoor coarse particles was conducted during the winter and summer of 2007. The coarse particle filters were subsequently analysed for endotoxin content to determine the contribution of this compound. Winter indoor geometric mean concentrations of coarse particles exceeded outdoor concentrations (3.73 μg m -3 vs 2.49 μg m -3; paired t-test p < 0.0001); however the reverse was found in summer (4.34 μg m -3 vs 8.82 μg m -3; paired t-test p < 0.0001). Linear regression indicated that winter predictors of indoor coarse particles were outdoor coarse particles, ventilation and presence of at least two or more occupants. During the summer, increased use of central air conditioning was associated with reduced coarse particles, while smoking and the presence of two or more occupants resulted in increased coarse particles. Endotoxin concentrations (EU μg -1) were lower indoors than outdoors in both seasons. Spatial variability of ambient coarse particles was assessed to determine the suitability of using a single monitoring station within a city to estimate exposure. The coefficients of variation between homes sampled simultaneously and the central monitoring station were calculated (median COV in summer = 15% and winter = 24%) and showed significant variability by week, especially during the summer months, suggesting a single site may be insufficient for characterizing exposure. Future studies should consider daily measurements per home to understand shorter term exposures and day to day

  14. Distribution and Kinetics of Lipoprotein-Bound Endotoxin

    PubMed Central

    Levels, J. H. M.; Abraham, P. R.; van den Ende, A.; van Deventer, S. J. H.

    2001-01-01

    Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent endotoxin responsible for pathophysiological symptoms characteristic of infection. The observation that the majority of LPS is found in association with plasma lipoproteins has prompted the suggestion that sequestering of LPS by lipid particles may form an integral part of a humoral detoxification mechanism. Previous studies on the biological properties of isolated lipoproteins used differential ultracentrifugation to separate the major subclasses. To preserve the integrity of the lipoproteins, we have analyzed the LPS distribution, specificity, binding capacity, and kinetics of binding to lipoproteins in human whole blood or plasma by using high-performance gel permeation chromatography and fluorescent LPS of three different chemotypes. The average distribution of O111:B4, J5, or Re595 LPS in whole blood from 10 human volunteers was 60% (±8%) high-density lipoprotein (HDL), 25% (±7%) low-density lipoprotein, and 12% (±5%) very low density lipoprotein. The saturation capacity of lipoproteins for all three LPS chemotypes was in excess of 200 μg/ml. Kinetic analysis however, revealed a strict chemotype dependence. The binding of Re595 or J5 LPS was essentially complete within 10 min, and subsequent redistribution among the lipoprotein subclasses occurred to attain similar distributions as O111:B4 LPS at 40 min. We conclude that under simulated physiological conditions, the binding of LPS to lipoproteins is highly specific, HDL has the highest binding capacity for LPS, the saturation capacity of lipoproteins for endotoxin far exceeds the LPS concentrations measured in clinical situations, and the kinetics of LPS association with lipoproteins display chemotype-dependent differences. PMID:11292694

  15. Ocular Albumin Fluorophotometric Quantitation of Endotoxin-Induced Vascular Permeability

    PubMed Central

    Cousins, Scott W.; Rosenbaum, James T.; Guss, Robert B.; Egbert, Peter R.

    1982-01-01

    Bacterial endotoxin (lipopolysaccharide; LPS) is known to alter systemic vascular permeability, but this effect is difficult to monitor and quantitate in vivo. The ocular vessels of the rabbit are particularly sensitive to LPS. Using a slit lamp equipped with a fluorophotometer, we have adapted a method to quantitate endotoxin-induced ocular vascular permeability by measuring the accumulation of fluorescein isothiocyanate-conjugated albumin into the anterior chamber of the eye. After intravenous administration of Salmonella typhimurim LPS, the anterior chamber fluorescence and blood fluorescence were measured at intervals of 15 min and 1 h, respectively, over 4 h. In controls, maximal fluorescence in the anterior chamber was 3.1 ± 0.8% of blood fluorescence. Doses of LPS as low as 0.25 μg/kg produced an ocular/serum fluorescence ratio of 17.6 ± 4.9. A dose of 2.5 μg of LPS per kg tended to produce a higher ratio (68.0 ± 7.1) than a larger dose of 50 μg/kg (30.5 ± 16.6). Permeability changes began within 30 min after LPS, and the rate of dye accumulation varied over time, with maximal leakage usually occurring 90 min after LPS, but occasionally occurring much later. Repeated doses produced tolerance. By conjugating albumin to rhodamine and utilizing a second filter with the slit lamp to measure accumulation of this dye, we demonstrated the persistence of marked permeability during a period when intraocular fluorescein isothiocyanate and albumin levels were relatively constant. This methodology indicates that extremely low doses of LPS induce ocular permeability changes and that neither the time course nor the dose response of this effect is linear. Ocular fluorophotometry is a sensitive, noninvasive technique to study the dynamics and pharmacology of LPS-induced permeability changes. PMID:6806194

  16. Hemoglobin: a newly recognized binding protein for bacterial endotoxins (LPS).

    PubMed

    Roth, R I; Kaca, W; Levin, J

    1994-01-01

    Administration of purified hemoglobin (Hb) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of Hb, we examined mixtures of Hb and LPS for evidence of LPS-Hb complex formation. LPS-Hb complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through sucrose, which distinguished denser LPS alone from LPS-Hb complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of Hb in complexes compared to Hb alone. Interaction of LPS with Hb was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several of the physical characteristics of LPS and enhancement of the biological activities of LPS. These findings suggest that a mechanism for the toxicity of infused Hb in vivo may involve potentiation of the biological effects of LPS. In addition, these observations suggest a mechanism by which LPS-related morbidity during sepsis could be enhanced by erythrocyte hemolysis.

  17. Gender Difference in Bacteria Endotoxin-Induced Inflammatory and Anorexic Responses.

    PubMed

    Kuo, Shiu-Ming

    2016-01-01

    Inflammation-related anorexic response has been observed in systemic diseases as well as in localized infection and is an important issue in patient care. We tested the hypothesis that upon the same endotoxin exposure, males have more severe inflammatory responses and thus suffer from more negative effect on appetite. Ten-week old male and female mice were compared in their plasma levels of pro-inflammatory cytokines after a body weight-based i.p. injection of bacterial endotoxin lipopolysaccharide. Male mice consistently showed significantly higher levels of IL6 and TNFα than female mice. The difference was observed starting at 3 hours after the systemic endotoxin exposure. It was independent of the level of endotoxin dosage and of the genotype of the anti-inflammatory cytokine, IL10. Interestingly, endotoxin-injected male mice also had significantly higher plasma IL10 levels compared to the female mice. Pre-puberty young mice showed no gender differences in the plasma levels of IL6, TNFα and IL10. Their cytokine levels were mostly between that of the adult males and females. Consistent with the higher inflammatory response in male mice, the endotoxin exposure also led to significantly more appetite loss in male mice at a range of doses in two strains of mice. Saline injection in the absence of endotoxin affected neither the cytokine levels nor the appetite. Although a direct mechanistic link between inflammation parameters and appetite was not addressed here, the results support that male gender could be a risk factor for higher pro-inflammatory cytokines and anorexic response after the endotoxin exposure. PMID:27631979

  18. Electrostatic dust collectors compared to inhalable samplers for measuring endotoxin concentrations in farm homes.

    PubMed

    Kilburg-Basnyat, B; Peters, T M; Perry, S S; Thorne, P S

    2016-10-01

    Paired electrostatic dust collectors (EDCs) and daily, inhalable button samplers (BS) were used concurrently to sample endotoxin in 10 farm homes during 7-day periods in summer and winter. Winter sampling included an optical particle counter (OPC) to measure PM2.5 and PM2.5-10 . Electrostatic dust collectors and BS filters were analyzed for endotoxin using the kinetic chromogenic Limulus amebocyte lysate assay. Optical particle counter particulate matter (PM) data were divided into two PM categories. In summer, geometric mean (geometric standard deviation) endotoxin concentrations were 0.82 EU/m(3) (2.7) measured with the BS and 737 EU/m(2) (1.9) measured with the EDC. Winter values were 0.52 EU/m(3) (3.1) for BS and 538 EU/m(2) (3.0) for EDCs. Seven-day endotoxin values of EDCs were highly correlated with the 7-day BS sampling averages (r = 0.70; P < 0.001). Analysis of variance indicated a 2.4-fold increase in EDC endotoxin concentrations for each unit increase of the ratio of PM2.5 to PM2.5-10 . There was also a significant correlation between BS and EDCs endotoxin concentrations for winter (r = 0.67; P < 0.05) and summer (r = 0.75; P < 0.05). Thus, EDCs sample comparable endotoxin concentrations to BS, making EDCs a feasible, easy to use alternative to BS for endotoxin sampling.

  19. Gender Difference in Bacteria Endotoxin-Induced Inflammatory and Anorexic Responses

    PubMed Central

    2016-01-01

    Inflammation-related anorexic response has been observed in systemic diseases as well as in localized infection and is an important issue in patient care. We tested the hypothesis that upon the same endotoxin exposure, males have more severe inflammatory responses and thus suffer from more negative effect on appetite. Ten-week old male and female mice were compared in their plasma levels of pro-inflammatory cytokines after a body weight-based i.p. injection of bacterial endotoxin lipopolysaccharide. Male mice consistently showed significantly higher levels of IL6 and TNFα than female mice. The difference was observed starting at 3 hours after the systemic endotoxin exposure. It was independent of the level of endotoxin dosage and of the genotype of the anti-inflammatory cytokine, IL10. Interestingly, endotoxin-injected male mice also had significantly higher plasma IL10 levels compared to the female mice. Pre-puberty young mice showed no gender differences in the plasma levels of IL6, TNFα and IL10. Their cytokine levels were mostly between that of the adult males and females. Consistent with the higher inflammatory response in male mice, the endotoxin exposure also led to significantly more appetite loss in male mice at a range of doses in two strains of mice. Saline injection in the absence of endotoxin affected neither the cytokine levels nor the appetite. Although a direct mechanistic link between inflammation parameters and appetite was not addressed here, the results support that male gender could be a risk factor for higher pro-inflammatory cytokines and anorexic response after the endotoxin exposure. PMID:27631979

  20. Effect of radio-detoxified endotoxin on the liver microsomal drug metabolizing enzyme system in rats

    SciTech Connect

    Bertok, L.; Szeberenyi, S.

    1983-06-01

    E. coli endotoxin (LPS) depresses the hepatic microsomal mono-oxygenase activity. Radio-detoxified LPS (TOLERIN: /sup 60/Co irradiated endotoxin preparation) decreases this biotransforming activity to a smaller extent. Phenobarbital, an inducer of this mono-oxygenase system, failed to induce in LPS-treated animals. In radio-detoxified LPS-treated rats, phenobarbital induced the mono-oxygenase and almost fully restored the biotransformation.

  1. A non-chromatographic method for the removal of endotoxins from bacteriophages.

    PubMed

    Branston, Steven D; Wright, Jason; Keshavarz-Moore, Eli

    2015-08-01

    The Ff filamentous bacteriophages show potential as a new class of therapeutics, displaying utility in materials science as well as pharmaceutical applications. These phages are produced by the infection of E. coli, a Gram-negative bacterium which unavoidably sheds endotoxins into the extracellular space during growth. Since endotoxin molecules are highly immunoreactive, separation from the phage product is of critical importance, particularly those developed for human therapeutic use. The properties of M13, one of the Ff group, present a purification challenge chiefly because the standard scalable method for endotoxin removal from proteins-anion exchange chromatography-is not applicable due to pI similarity between the particles. This article examines the potential of polyethylene glycol (PEG)-NaCl precipitation as a scalable method for the separation of endotoxins from phage M13. Precipitation of M13 by 2% (w/v) PEG 6 000, 500 mM NaCl reduced endotoxin contamination of the phage product by 88%, but additional precipitation rounds did not maintain this proportional decrease. Dynamic light scattering was subsequently used to determine the effectiveness of a detergent to disassociate endotoxin molecules from M13. As a result, PEG-NaCl precipitation was supplemented with up to 2% (v/v) Triton X-100 to improve separation. A 5.7 log10 reduction in endotoxin concentration was achieved over three rounds of precipitation whilst retaining over 97% of the phage. This method compares favorably with the well-known ATPS (Triton X-114) technique for endotoxin removal from protein solutions.

  2. Understanding pesticides in California's Delta

    USGS Publications Warehouse

    Kuivila, Kathryn M.; Orlando, James L.

    2012-01-01

    The Sacramento-San Joaquin River Delta (Delta) is the hub of California’s water system and also an important habitat for imperiled fish and wildlife. Aquatic organisms are exposed to mixtures of pesticides that flow through the maze of Delta water channels from sources including agricultural, landscape, and urban pest-control applications. While we do not know all of the effects pesticides have on the ecosystem, there is evidence that they cause some damage to organisms in the Delta. Decades of USGS research have provided a good understanding of when, where, and how pesticides enter the Delta. However, pesticide use is continually changing. New field studies and methods are needed so that scientists can analyze which pesticides are present in the Delta, and at what concentrations, enabling them to estimate exposure and ultimate effects on organisms. Continuing research will provide resource managers and stakeholders with crucial information to manage the Delta wisely.

  3. Chasing a ghost?--Issues with the determination of circulating levels of endotoxin in human blood.

    PubMed

    Gnauck, Anne; Lentle, Roger Graham; Kruger, Marlena Cathorina

    2016-01-01

    Reliable quantification of bacterial products such as endotoxin is important for the diagnosis of Gram-negative infection and for the monitoring of its treatment. Further, it is important to identify patients with persistent subclinical level of bacterial products in their systemic circulation as data from animal studies also suggest this may be correlated with the onset of metabolic syndrome. In this review, we first aim to describe the principles of the Limulus amoebocyte lysate (LAL) test, an assay that is used to quantify endotoxin, and the various shortcomings that must be addressed before it can become a reliable means of quantifying endotoxin in samples derived from blood. We then review published data regarding endotoxin levels in healthy subjects and those with sepsis, inflammatory bowel disease, liver disorders and metabolic disorders such as obesity and diabetes. We also review the evidence regarding influence of macronutrients in augmenting the levels of systemic endotoxin. The results of this review show that reported mean levels of endotoxin in the systemic circulation of healthy humans and of those with various clinical disorders vary over a wide range. Further, this review shows that a significant proportion of this variation can be related to the method that was used to prepare plasma and serum samples prior to assay and its ability to reduce the effect of various blood borne factors that interfere with the LAL assay. PMID:26732012

  4. Comparison of Different Irrigants in the Removal of Endotoxins and Cultivable Microorganisms from Infected Root Canals

    PubMed Central

    Valera, Marcia Carneiro; Cardoso, Flávia Goulart da Rosa; Chung, Adriana; Xavier, Ana Cláudia Carvalho; Figueiredo, Mariana Diehl; Martinho, Frederico Canato; Palo, Renato Miotto

    2015-01-01

    This study was conducted to compare the effectiveness of different irrigants used to remove endotoxins and cultivable microorganisms during endodontic therapy. Forty root canals were contaminated and divided into groups according to the irrigant: 2% NaOCl + surfactant, 2% CHX, 2.5% NaOCl, and pyrogen-free saline solution (control). Samples were collected after root canal contamination (S1), after instrumentation (S2), and 7 days after instrumentation (S3). Microorganisms and endotoxins were recovered from 100% of the contaminated root canals (S1). At S2, 2% NaOCl + surfactant, 2% CHX, and 2.5% NaOCl were able to completely eliminate cultivable microorganisms. At S3, both 2% CHX and 2.5% NaOCl were effective in preventing C. albicans and E. coli regrowth, but E. faecalis was still detected. No microorganism species was recovered from root canals instrumented with 2% NaOCl + surfactant. At S2, a higher percentage value of endotoxin reduction was found for 2% NaOCl + surfactant (99.3%) compared to 2% CHX (98.9%) and 2.5% NaOCl (97.18%) (p < 0.05). Moreover, at S3, 2% NaOCl + surfactant (100%) was the most effective irrigant against endotoxins. All irrigants tested were effective in reducing microorganisms and endotoxins from root canals. Moreover, 2% NaOCl + surfactant was the most effective irrigant against endotoxins and regrowth of microorganisms. PMID:26346574

  5. Development of an electrochemical Limulus amebocyte lysate assay technique for portable and highly sensitive endotoxin sensor.

    PubMed

    Inoue, Kumi Y; Takahashi, Satoko; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

    2012-04-01

    Here, we report the development of an electrochemical detection method for endotoxin based on the Limulus amebocyte lysate (LAL) assay. A mixture of LAL reagent and endotoxin sample solution was incubated for 1 h. The endotoxin activated a cascade reaction of zymogens contained in the LAL to generate p-nitroaniline (pNA) which was then electrochemically detected by differential pulse voltammetry (DPV). The generated pNA gave a clear peak at -0.75 V vs. silver/silver chloride (Ag/AgCl), which increased with the concentration of endotoxin in the LAL assay solution. This DPV detection was performed using an electrode chip device fabricated from a diamond-like carbon-coated glass substrate. This chip device could detect as low as 10 endotoxin units l(-1) at room temperature within 1 h. This novel electrochemical method for the detection of endotoxin appears promising for the development of compact, low-cost and easy-to-use sensors for on-site monitoring of potentially contaminated medical supplies, including dialysis fluid, transplanted tissue and culture medium for assisted reproduction. PMID:21844129

  6. Endotoxin and β-(1,3)-glucan levels in automobiles: a pilot study.

    PubMed

    Wu, Francis Fu-Sheng; Wu, Mei-Wen; Chang, Chin-Fu; Lai, Shu-Mei; Pierse, Nevil; Crane, Julian; Siebers, Rob

    2010-01-01

    Exposure to bacterial endotoxin and fungal β-(1,3)-glucan in the indoor environment can induce respiratory symptoms. Automobiles are an exposure source of allergens but it is not known if, and how much exposure there is to endotoxin and fungal β-(1,3)-glucan. The objective of the study was to determine whether automobiles are a potential source of exposure to these microbial products. Dust was sampled from the passenger seats of 40 automobiles. Specific Limulus amoebocyte kinetic assays were used to measure endotoxin and β-(1,3)-glucan, respectively. Endotoxin and β-(1,3)-glucan was detected in all samples ranging from 19.9-247.0 EU/mg and 1.6-59.8 μg/g, respectively. There were no significant differences in endotoxin levels between automobiles of smokers and non-smokers, but β-(1,3)-glucan levels were about two-fold higher in the automobiles of non-smokers. In conclusion, endotoxin and β-(1,3)-glucan exposure in automobiles at levels found in our study may be of importance for asthmatics.

  7. Human endotoxin tolerance is associated with enrichment of the CD14+ CD16+ monocyte subset.

    PubMed

    Domínguez-Nieto, Aimée; Zentella, Alejandro; Moreno, José; Ventura, José L; Pedraza, Sigifredo; Velázquez, Juan R

    2015-01-01

    Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C-X-C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine.

  8. Human endotoxin tolerance is associated with enrichment of the CD14+ CD16+ monocyte subset.

    PubMed

    Domínguez-Nieto, Aimée; Zentella, Alejandro; Moreno, José; Ventura, José L; Pedraza, Sigifredo; Velázquez, Juan R

    2015-01-01

    Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C-X-C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine. PMID:25172544

  9. Comparison of Different Irrigants in the Removal of Endotoxins and Cultivable Microorganisms from Infected Root Canals.

    PubMed

    Valera, Marcia Carneiro; Cardoso, Flávia Goulart da Rosa; Chung, Adriana; Xavier, Ana Cláudia Carvalho; Figueiredo, Mariana Diehl; Martinho, Frederico Canato; Palo, Renato Miotto

    2015-01-01

    This study was conducted to compare the effectiveness of different irrigants used to remove endotoxins and cultivable microorganisms during endodontic therapy. Forty root canals were contaminated and divided into groups according to the irrigant: 2% NaOCl + surfactant, 2% CHX, 2.5% NaOCl, and pyrogen-free saline solution (control). Samples were collected after root canal contamination (S1), after instrumentation (S2), and 7 days after instrumentation (S3). Microorganisms and endotoxins were recovered from 100% of the contaminated root canals (S1). At S2, 2% NaOCl + surfactant, 2% CHX, and 2.5% NaOCl were able to completely eliminate cultivable microorganisms. At S3, both 2% CHX and 2.5% NaOCl were effective in preventing C. albicans and E. coli regrowth, but E. faecalis was still detected. No microorganism species was recovered from root canals instrumented with 2% NaOCl + surfactant. At S2, a higher percentage value of endotoxin reduction was found for 2% NaOCl + surfactant (99.3%) compared to 2% CHX (98.9%) and 2.5% NaOCl (97.18%) (p < 0.05). Moreover, at S3, 2% NaOCl + surfactant (100%) was the most effective irrigant against endotoxins. All irrigants tested were effective in reducing microorganisms and endotoxins from root canals. Moreover, 2% NaOCl + surfactant was the most effective irrigant against endotoxins and regrowth of microorganisms.

  10. Experimental Study on Inactivation of Bacterial Endotoxin by Using Dielectric Barrier Discharge

    NASA Astrophysics Data System (ADS)

    Shi, Xingmin; Li, Yaxi; Zhang, Guanjun; Ma, Yue; Shao, Xianjun

    2011-12-01

    The low-temperature plasma (LTP) generated by dielectric barrier discharge (DBD) was used to sterilize the E.coli endotoxin, which is usually difficult to kill by traditional methods. Three different concentrations of bacterial endotoxin (1 EU/mL, 0.5 EU/mL and 0.25 EU/mL) were treated by LTP for different time (20 s, 40 s and 60 s). Tachypleus amebocyte lysate (TAL) method was employed to detect the concentration variation of bacterial endotoxin before and after the plasma treatment, and endotoxic shock mice model was used to evaluate the inactivation effects of LTP on endotoxin for further study. Experimental results demonstrated that, DBD plasma can inactivate the bacterial endotoxin quickly and effectively, and when the LTP treatment time was increased, the concentrations of bacterial endotoxin decreased gradually (after 60 s plasma treatment, its inactivation effect was beyond the Chinese pharmacopoeia standard), and the average survival time of mice gradually extended. The possible inactivation mechanisms are proposed to be related to reactive oxygen species (ROSs).

  11. Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

    SciTech Connect

    Not Available

    1986-01-05

    The effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells were examined. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner. The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP)-abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56/sup 0/C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of /sup 125/I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa.

  12. Removal of endotoxin from deionized water using micromachined silicon nanopore membranes

    NASA Astrophysics Data System (ADS)

    Smith, Ross A.; Goldman, Ken; Fissell, William H.; Fleischman, Aaron J.; Zorman, Christian A.; Roy, Shuvo

    2011-05-01

    Endotoxins are lipopolysaccharide components of the cell membrane of Gram-negative bacteria that trigger the body's innate immune system and can cause shock and death. Water for medical therapy, including parenteral and dialysate solutions, must be free of endotoxin. This purity is challenging to achieve as many Gram-negative bacteria are endemic in the environment, and can thrive in harsh, nutrient-poor conditions. Current methods for removing endotoxin include distillation and reverse osmosis, both of which are resource intensive processes. Membranes that present an absolute barrier to macromolecular passage may be capable of delivering pure water for biomedical applications. In this work, endotoxin has been filtered from aqueous solutions using silicon nanopore membranes (SNMs) with monodisperse pore size distributions. SNMs with critical pore sizes between 26 and 49 nm were challenged with solutions of deionized water spiked with endotoxin and with Pseudomonas cepacia. The filtrate produced by the SNM from Pseudomonas-contaminated water had <1.0 endotoxin unit (EU) ml-1, which meets standards for dialysate purity. This approach suggests a technique for single-step cleanup of heavily contaminated water that may be suitable for field or clinical use.

  13. Altered macrophage arachidonic acid metabolism induced by endotoxin tolerance: characterization and mechanisms

    SciTech Connect

    Rogers, T.S.

    1986-01-01

    Altered macrophage arachidonic acid (AA) metabolism may play a role in endotoxic shock and the phenomenon of endotoxin tolerance induced by repeated injections of endotoxin. Studies were initiated to characterize both lipoxygenase and cyclooxygenase metabolite formation by endotoxin tolerant and non-tolerant macrophages in response to 4 different stimuli, i.e., endotoxin, glucan, zymosan, and the calcium ionophore A23187. In contrast to previous reports of decreased prostaglandin synthesis by tolerant macrophages, A23187-stimulated immunoreactive (i) leukotriene (LT) C/sub 4/D/sub 4/ and prostaglandin (PG) E/sub 2/ production by tolerant cells was greater than that by non-tolerant controls (p <0.001). However, A23187-stimulated i6-keto PGF/sub 1a/ levels were lower in tolerant macrophages compared to controls (P < 0.05). iL TC/sub 4/D/sub 4/ production was not significantly stimulated by endotoxin or glucan, but was stimulated by zymosan in non-tolerant cells. Synthesis of iLTB/sub 4/ by control macrophages was stimulated by endotoxin (p <0.01). The effect of tolerance on factors that affect AA release was investigated by measuring /sup 14/C-AA incorporation and release and phospholipase A/sub 2/ activity

  14. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

    PubMed

    Schwarz, Harald; Schmittner, Maria; Duschl, Albert; Horejs-Hoeck, Jutta

    2014-01-01

    Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

  15. Polymyxin B immobilized on cross-linked cellulose microspheres for endotoxin adsorption.

    PubMed

    Cao, Xiaodong; Zhu, Biyan; Zhang, Xufeng; Dong, Hua

    2016-01-20

    Cross-linked cellulose microspheres (CL-CMs) were successfully prepared by inverse crosslinking suspension method. NaOH/urea aqueous solution was used as solvent to dissolve cellulose at low temperature. The microspheres presented good spherical shape and monodispersity, which were applied to synthesize endotoxin adsorbent with polymyxin B (PMB) as ligand. The adsorbent showed good adsorption capability on endotoxin in physiologic saline solution and the maximum adsorption capacity was 3605 EU/g (1 EU=100 pg). It was worth noting that more than 70% of endotoxin could be effectively removed from the human plasma with the initial concentration of endotoxin ranged from 1 EU/mL to 5 EU/mL. The dynamic adsorption efficiency of endotoxin was 72.3% at the plasma perfusion rate of 300 mL/h with the endotoxin concentration of 4 EU/mL, while the variation of plasma protein before and after adsorption was only 8.9%. It suggests that the PMB immobilized CL-CMs have great potential application in clinical blood purification.

  16. Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

    PubMed

    Szermer-Olearnik, Bożena; Boratyński, Janusz

    2015-01-01

    Lipopolysaccharide (LPS, endotoxin, pyrogen) constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol). During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU) in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 10(3) and 10(5) EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 10(3)-10(5) EU/10(9) PFU (plaque forming units) down to an average of 2.8 EU/10(9) PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa).

  17. Tumor necrosis factor and interleukin 1 as mediators of endotoxin-induced beneficial effects

    SciTech Connect

    Urbaschek, R.; Urbaschek, B.

    1987-09-01

    Bacterial lipopolysaccharides or endotoxins are known to induce tumor necrosis; enhanced nonspecific resistance to bacterial, viral, and parasitic infections and to radiation sickness; and tolerance to lethal doses of endotoxin. These beneficial effects are achieved by pretreatment with minute amounts of endotoxin. Recombinant tumor necrosis factor (TNF) and interleukin 1 (IL-1) are among the mediators capable of invoking radioprotection or resistance to the consequences of cecal ligation and puncture. Both cytokines are potent inducers of serum colony-stimulating factor (CSF) in C3H/HeJ mice (low responders to endotoxin). The number of splenic granulocyte-macrophage precursors was found to increase 5 days after injection of TNF in these mice. Although with IL-1 no increase in the number of granulocyte-macrophage colonies occurred in culture in the presence of serum CSF, a marked stimulation was observed when TNF was added. This stimulation of myelopoiesis observed in vivo and in vitro may be related to the radioprotective effect of TNF. The data presented suggest that TNF and IL-1 released after injection of endotoxin participate in the mediation of endotoxin-induced enhancement of nonspecific resistance and stimulation of hematopoiesis. 76 references.

  18. Relationship between endotoxin and prostaglandin (PGE2 and PGFM) concentrations and ovarian function in dairy cows with puerperal endometritis.

    PubMed

    Mateus, L; Lopes da Costa, L; Diniz, P; Ziecik, A J

    2003-04-15

    Blood concentrations of progesterone, 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM) and endotoxin, and uterine fluid concentrations of prostaglandin E(2) (PGE(2)), PGFM and endotoxin were evaluated in 14 dairy cows with puerperal endometritis (mild (n=6) and heavy (n=8)). Endotoxin was measured using a quantitative kinetic assay. Cows with heavy endometritis had significantly higher concentrations of plasma PGFM (P<0.01) and uterine fluid PGE(2) and endotoxin (P<0.05) than cows with mild endometritis. Concentrations of PGFM in plasma and uterine fluid, of PGFM and PGE(2), and PGE(2) and endotoxin in uterine fluid were positively and significantly (P<0.05) correlated. The presence of endotoxin in plasma was detected in one out of six mild and in eight out of eight heavy endometritis cows. Peak plasma endotoxin concentrations (0.08-9.14 endotoxin units/ml (EU/ml) were observed between 1 and 12 days postpartum (pp) and thereafter amounts generally remained below 0.1 EU/ml (last day of detection: Day 27 pp). Abnormal ovarian function was observed in six cows (four with prolonged anoestrus and two with long luteal phase after the first postpartum ovulation). Plasma endotoxin concentrations were detected in the anoestric cows. The results suggest that: (i) concentrations of uterine fluid endotoxin and PGE(2) and of plasma PGFM are related to the degree of endometritis; (ii) absorption of endotoxin from the uterus to the bloodstream occurs, mainly in heavy endometritis cows; and (iii) there is a relationship between uterine infection, endotoxin production and resumption of pp ovarian activity. PMID:12586488

  19. Mississippi River Delta

    NASA Technical Reports Server (NTRS)

    2002-01-01

    As the Mississippi River enters the Gulf of Mexico, it loses energy and dumps its load of sediment that it has carried on its journey through the mid continent. This pile of sediment, or mud, accumulates over the years building up the delta front. As one part of the delta becomes clogged with sediment, the delta front will migrate in search of new areas to grow. The area shown on this image is the currently active delta front of the Mississippi. The migratory nature of the delta forms natural traps for oil. Most of the land in the image consists of mud flats and marsh lands. There is little human settlement in this area due to the instability of the sediments. The main shipping channel of the Mississippi River is the broad stripe running northwest to southeast.

    This image was acquired on May 24, 2001 by the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) on NASA's Terra satellite. With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER will image Earth for the next 6 years to map and monitor the changing surface of our planet.

    ASTER is one of five Earth-observing instruments launched December 18,1999, on NASA's Terra satellite. The instrument was built by Japan's Ministry of Economy, Trade and Industry. A joint U.S./Japan science team is responsible for validation and calibration of the instrument and the data products. Dr. Anne Kahle at NASA's Jet Propulsion Laboratory, Pasadena, California, is the U.S. Science team leader; Bjorn Eng of JPL is the project manager. The Terra mission is part of NASA's Earth Science Enterprise, a long-term research and technology program designed to examine Earth's land, oceans, atmosphere, ice and life as a total integrated system.

    The broad spectral coverage and high spectral resolution of ASTER will provide scientists in numerous disciplines with critical information for surface mapping

  20. Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

    PubMed

    Sotohira, Yukari; Suzuki, Kazuyuki; Sasaki, Haruka; Sano, Tadashi; Tsuchiya, Masakazu; Suzuki, Yohko; Shimamori, Toshio; Tsukano, Kenji; Sato, Ayano; Yokota, Hiroshi; Asakawa, Mitsuhiko

    2016-07-01

    The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTS(TM)) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assay. The data obtained using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001). These findings indicated that the PTS(TM) is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods.

  1. Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system

    PubMed Central

    SOTOHIRA, Yukari; SUZUKI, Kazuyuki; SASAKI, Haruka; SANO, Tadashi; TSUCHIYA, Masakazu; SUZUKI, Yohko; SHIMAMORI, Toshio; TSUKANO, Kenji; SATO, Ayano; YOKOTA, Hiroshi; ASAKAWA, Mitsuhiko

    2016-01-01

    The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTSTM) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTSTM was not significantly different from that of the traditional LAL-based assay. The data obtained using PTSTM correlated with those using KT (r2=0.963, P<0.001). These findings indicated that the PTSTM is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods. PMID:26902804

  2. Endotoxin Detection in Pharmaceuticals and Medical Devices with Kinetic-QCL, a Kinetic-Quantitative Chromogenic Limulus Amebocyte Lysate Assay.

    PubMed

    Berzofsky, Ronald N.

    1995-01-01

    The observation that endotoxin caused gelation in extracts of Limulus amebocytes has been expanded to the development of an in vitro kinetic, quantitative chromogenic LAL assay (Kinetic-QCL) for the detection of endotoxin in aqueous fluids. Within the last 15 years, the use of Limulus amebocyte lysate to detect and control the presence of pyrogenic substances in pharmaceuticals and medical devices has gained wide international acceptance. Both the United States and European Pharmacopoeias contain descriptions of and requirements for the LAL Bacterial Endotoxin Test. Both pharmacopoeias have begun to remove the rabbit pyrogen test requirement in a majority of drug monographs and have substituted endotoxin limits to be determined by LAL. The use of LAL has proved invaluable in controlling the level of endotoxin in finished product. The endotoxin contribution of raw materials and packaging material can be monitored as well. In-process testing at critical production steps can identify additional sources of endotoxin contamination, and depyrogenation processes can be validated by quantitating the degradation of endotoxin challenges. The speed, reproducibility, sensitivity, and economics of the Kinetic-QCL assay, in conjunction with the ppropriate equipment and software, over both the in vivo rabbit pyrogen test and the more traditional LAL gel-clot assay allow a more in-depth approach to the control of endotoxin in pharmaceuticals and medical devices.

  3. Plasma endotoxin activity in kangaroos with oral necrobacillosis (lumpy jaw disease) using an automated handheld testing system.

    PubMed

    Sotohira, Yukari; Suzuki, Kazuyuki; Sasaki, Haruka; Sano, Tadashi; Tsuchiya, Masakazu; Suzuki, Yohko; Shimamori, Toshio; Tsukano, Kenji; Sato, Ayano; Yokota, Hiroshi; Asakawa, Mitsuhiko

    2016-07-01

    The aim of the present study was to evaluate the reliability and effectiveness of directly determining endotoxin activity in plasma samples from kangaroos with lumpy jaw disease (LJD, n=15) and healthy controls (n=12). Prior to the present study, the ability of the commercially available automated handheld portable test system (PTS(TM)) to detect endotoxin activity in kangaroo plasma was compared with that of the traditional LAL-kinetic turbidimetric (KT) assay. Plasma samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTS(TM) was not significantly different from that of the traditional LAL-based assay. The data obtained using PTS(TM) correlated with those using KT (r(2)=0.963, P<0.001). These findings indicated that the PTS(TM) is applicable as a simplified system to assess endotoxin activity in macropods. In the present study, we demonstrated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with LJD. Based on ROC curves, we proposed a diagnostic cut-off point for endotoxin activity of >0.22 EU/ml for the identification of LJD. Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for determining the outcome of LJD in captive macropods. PMID:26902804

  4. Natural processes in delta restoration: application to the Mississippi Delta.

    PubMed

    Paola, Chris; Twilley, Robert R; Edmonds, Douglas A; Kim, Wonsuck; Mohrig, David; Parker, Gary; Viparelli, Enrica; Voller, Vaughan R

    2011-01-01

    Restoration of river deltas involves diverting sediment and water from major channels into adjoining drowned areas, where the sediment can build new land and provide a platform for regenerating wetland ecosystems. Except for local engineered structures at the points of diversion, restoration mainly relies on natural delta-building processes. Present understanding of such processes is sufficient to provide a basis for determining the feasibility of restoration projects through quantitative estimates of land-building rates and sustainable wetland area under different scenarios of sediment supply, subsidence, and sea-level rise. We are not yet to the point of being able to predict the evolution of a restored delta in detail. Predictions of delta evolution are based on field studies of active deltas, deltas in mine-tailings ponds, experimental deltas, and countless natural experiments contained in the stratigraphic record. These studies provide input for a variety of mechanistic delta models, ranging from radially averaged formulations to more detailed models that can resolve channels, topography, and ecosystem processes. Especially exciting areas for future research include understanding the mechanisms by which deltaic channel networks self-organize, grow, and distribute sediment and nutrients over the delta surface and coupling these to ecosystem processes, especially the interplay of topography, network geometry, and ecosystem dynamics. PMID:21329199

  5. Immobilization of ɛ-polylysine onto the probe surface for molecular adsorption type endotoxin detection system

    NASA Astrophysics Data System (ADS)

    Ooe, Katsutoshi; Tsuji, Akihito; Nishishita, Naoki; Hirano, Yoshiaki

    2007-04-01

    Patients with renal failure become not able to expel the waste product, and they accumulate the toxic products for themselves. They therefore must use the hemodialysis to weed out the metabolic decomposition product. Hemodialysis for chronic renal failure is the most popular treatment method with artificial organs. However, hemodialysis patients must continue the treatment throughout their life, the results of long term extracorporeal dialysis, those patients develop the various complications and diseases, for example, dialysis amyloidosis etc. Dialysis amyloidosis is one of the refractory complications, and the prevention of this complication is important. Recently, endotoxin is thought to be the most likely cause of the complication. Endotoxin is one of the major cell wall components of gram-negative bacteria, and it forms the complex with proteins and lipopolysaccharide (LPS). It has various biological activities, e.g. attack of fever, when it gets mixed into human blood. In addition, it is known that large amount of endotoxin exists in living environment, and medicine is often contaminated with endotoxin. When contaminated dialyzing fluids are used to hemodialysis, above-mentioned dialysis amyloidosis is developed. Therefore, it is important that the detection and removal of endotoxin from dialyzing fluids. Until now, the measurement methods using Limulus Amebosyte Lysate (LAL) reagent were carried out as the tests for the presence of endotoxin. However, these methods include several different varieties of measurement techniques. The following are examples of them, gelatinization method, turbidimetric assay method, colorimetric assay method and fluoroscopic method. However, these techniques needed 30-60 minutes for the measurement. From these facts, they are not able to use as a "real-time endotoxin detector". The detection of endotoxin has needed to carry out immediately, for that reason, a new "real-time" detection method is desired. We focused attention to

  6. Pioneer Launch on Delta Vehicle

    NASA Technical Reports Server (NTRS)

    1969-01-01

    NASA launches the last in the series of interplanetary Pioneer spacecraft, Pioneer 10 from Cape Kennedy, Florida. The long-tank Delta launch vehicle placed the spacecraft in a solar orbit along the path of Earth's orbit. The spacecraft then passed inside and outside Earth's orbit, alternately speeding up and slowing down relative to Earth. The Delta launch vehicle family started development in 1959. The Delta was composed of parts from the Thor, an intermediate-range ballistic missile, as its first stage, and the Vanguard as its second. The first Delta was launched from Cape Canaveral on May 13, 1960 and was powerful enough to deliver a 100-pound spacecraft into geostationary transfer orbit. Delta has been used to launch civil, commercial, and military satellites into orbit. For more information about Delta, please see Chapter 3 in Roger Launius and Dennis Jenkins' book To Reach the High Frontier published by The University Press of Kentucky in 2002.

  7. Delta in Eberswalde

    NASA Technical Reports Server (NTRS)

    2006-01-01

    This HiRISE image covers a portion of a delta that partially fills Eberswalde crater in Margaritifer Sinus. The delta was first recognized and mapped using MOC images that revealed various features whose presence required sustained flow and deposition into a lake that once occupied the crater. The HiRISE image resolves meter-scale features that record the migration of channels and delta distributaries as the delta grew over time. Differences in grain-size of sediments within the environments on the delta enable differential erosion of the deposits. As a result, coarser channel deposits are slightly more resistant and stand in relief relative to finer-grained over-bank and more easily eroded distal delta deposits. Close examination of the relict channel deposits confirms the presence of some meter-size blocks that were likely too coarse to have been transported by water flowing within the channels. These blocks may be formed of the sand and gravel that more likely moved along the channels that was lithified and eroded. Numerous meter-scale polygonal structures are common on many surfaces, but mostly those associated with more quiescent depositional environments removed from the channels. The polygons could be the result of deposition of fine-grained sediments that were either exposed and desiccated (dried out), rich in clays that shrunk when the water was removed, turned into rock and then fractured and eroded, or some combination of these processes.

    Image PSP_001336_1560 was taken by the High Resolution Imaging Science Experiment (HiRISE) camera onboard the Mars Reconnaissance Orbiter spacecraft on November 8, 2006. The complete image is centered at -23.8 degrees latitude, 326.4 degrees East longitude. The range to the target site was 256.3 km (160.2 miles). At this distance the image scale is 25.6 cm/pixel (with 1 x 1 binning) so objects 77 cm across are resolved. The image shown here has been map-projected to 25 cm/pixel and north is up. The image was

  8. Diagnostic potential of endotoxin scattering photometry for sepsis and septic shock.

    PubMed

    Shimizu, Tomoharu; Obata, Toru; Sonoda, Hiromichi; Akabori, Hiroya; Miyake, Tohru; Yamamoto, Hiroshi; Tabata, Takahisa; Eguchi, Yutaka; Tani, Tohru

    2013-12-01

    Endotoxin scattering photometry (ESP) is a novel Limulus amebocyte lysate (LAL) assay that uses a laser light-scattering particle-counting method. In the present study, we compared ESP, standard turbidimetric LAL assay, and procalcitonin assay for the evaluation of sepsis after emergency gastrointestinal surgery. A total of 174 samples were collected from 40 adult patients undergoing emergency gastrointestinal surgery and 10 patients with colorectal cancer undergoing elective surgery as nonseptic controls. Plasma endotoxin levels were measured with ESP and turbidimetric LAL assay, and plasma procalcitonin levels were assessed with a standard procalcitonin assay. Plasma endotoxin and procalcitonin levels increased corresponding to the degree of sepsis. Endotoxin scattering photometry significantly discriminated between patients with or without septic shock: sensitivity, 81.1%; specificity, 76.6%; positive predictive value, 48.4%; negative predictive value, 93.8%; and accuracy, 77.6%. The area under the receiver operating characteristic curve for septic shock with the ESP assay (endotoxin cutoff value, 23.8 pg/mL) was 0.8532 ± 0.0301 (95% confidence interval, 0.7841-0.9030; P < 0.0001). The predictive power of ESP was superior to that of turbidimetric assay (difference, 0.1965 ± 0.0588; 95% confidence interval, 0.0812-0.3117; P = 0.0008). There was no significant difference in predictive power between ESP and procalcitonin assay. Endotoxin scattering photometry also discriminated between patients with and without sepsis. Area under the receiver operating characteristic curve analysis showed that ESP had the best predictive power for diagnosing sepsis. In conclusion, compared with turbidimetric LAL assay, ESP more sensitively detected plasma endotoxin and significantly discriminated between sepsis and septic shock in patients undergoing gastrointestinal emergency surgery.

  9. Long-term interdisciplinary therapy reduces endotoxin level and insulin resistance in obese adolescents

    PubMed Central

    2012-01-01

    Aim The purpose of the present study was to assess the dietary fat intake, glucose, insulin, Homeostasis model assessment for insulin resistance HOMA-IR, and endotoxin levels and correlate them with adipokine serum concentrations in obese adolescents who had been admitted to long-term interdisciplinary weight-loss therapy. Design The present study was a longitudinal clinical intervention of interdisciplinary therapy. Adolescents (n = 18, aged 15–19 y) with a body mass index > 95th percentile were admitted and evaluated at baseline and again after 1 year of interdisciplinary therapy. We collected blood samples, and IL-6, adiponectin, and endotoxin concentrations were measured by ELISA. Food intake was measured using 3-day diet records. In addition, we assessed glucose and insulin levels as well as the homeostasis model assessment for insulin resistance (HOMA-IR). Results The most important finding from the present investigation was that the long-term interdisciplinary lifestyle therapy decreased dietary fat intake and endotoxin levels and improved HOMA-IR. We observed positive correlations between dietary fat intake and endotoxin levels, insulin levels, and the HOMA-IR. In addition, endotoxin levels showed positive correlations with IL-6 levels, insulin levels and the HOMA-IR. Interestingly, we observed a negative correlation between serum adiponectin and both dietary fat intake and endotoxin levels. Conclusions The present results indicate an association between dietary fat intake and endotoxin level, which was highly correlated with a decreased pro-inflammatory state and an improvement in HOMA-IR. In addition, this benefits effect may be associated with an increased adiponectin level, which suggests that the interdisciplinary therapy was effective in improving inflammatory pathways. PMID:22989045

  10. Airborne environmental endotoxin: a cross-validation of sampling and analysis techniques.

    PubMed Central

    Walters, M; Milton, D; Larsson, L; Ford, T

    1994-01-01

    A standard method for measurement of airborne environmental endotoxin was developed and field tested in a fiberglass insulation-manufacturing facility. This method involved sampling with a capillary-pore membrane filter, extraction in buffer using a sonication bath, and analysis by the kinetic-Limulus assay with resistant-parallel-line estimation (KLARE). Cross-validation of the extraction and assay method was performed by comparison with methanolysis of samples followed by 3-hydroxy fatty acid (3-OHFA) analysis by gas chromatography-mass spectrometry. Direct methanolysis of filter samples and methanolysis of buffer extracts of the filters yielded similar 3-OHFA content (P = 0.72); the average difference was 2.1%. Analysis of buffer extracts for endotoxin content by the KLARE method and by gas chromatography-mass spectrometry for 3-OHFA content produced similar results (P = 0.23); the average difference was 0.88%. The source of endotoxin was gram-negative bacteria growing in recycled washwater used to clean the insulation-manufacturing equipment. The endotoxin and bacteria become airborne during spray cleaning operations. The types of 3-OHFAs in bacteria cultured from the washwater, present in the washwater and in the air, were similar. Virtually all of the bacteria cultured from air and water were gram negative composed mostly of two species, Deleya aesta and Acinetobacter johnsonii. Airborne countable bacteria correlated well with endotoxin (r2 = 0.64). Replicate sampling showed that results with the standard sampling, extraction, and Limulus assay by the KLARE method were highly reproducible (95% confidence interval for endotoxin measurement +/- 0.28 log10). These results demonstrate the accuracy, precision, and sensitivity of the standard procedure proposed for airborne environmental endotoxin. PMID:8161191

  11. Comparison of endotoxin and particle bounce in Marple cascade samplers with and without impaction grease.

    PubMed

    Kirychuk, Shelley P; Reynolds, Stephen J; Koehncke, Niels; Nakatsu, J; Mehaffy, John

    2009-01-01

    The health of persons engaged in agricultural activities are often related or associated with environmental exposures in their workplace. Accurately measuring, analyzing, and reporting these exposures is paramount to outcomes interpretation. This paper describes issues related to sampling air in poultry barns with a cascade impactor. Specifically, the authors describe how particle bounce can affect measurement outcomes and how the use of impaction grease can impact particle bounce and laboratory analyses such as endotoxin measurements. This project was designed to (1) study the effect of particle bounce in Marple cascade impactors that use polyvinyl chloride (PVC) filters; (2) to determine the effect of impaction grease on endotoxin assays when sampling poultry barn dust. A pilot study was undertaken utilizing six-stage Marple cascade impactors with PVC filters. Distortion of particulate size distributions and the effects of impaction grease on endotoxin analysis in samples of poultry dust distributed into a wind tunnel were studied. Although there was no significant difference in the overall dust concentration between utilizing impaction grease and not, there was a greater than 50% decrease in the mass median aerodynamic diameter (MMAD) values when impaction grease was not utilized. There was no difference in airborne endotoxin concentration or endotoxin MMAD between filters treated with impaction grease and those not treated. The results indicate that particle bounce should be a consideration when sampling poultry barn dust with Marple samplers containing PVC filters with no impaction grease. Careful consideration should be given to the utilization of impaction grease on PVC filters, which will undergo endotoxin analysis, as there is potential for interference, particularly if high or low levels of endotoxin are anticipated. PMID:19437284

  12. Association of endotoxins and colon polyp: a case-control study.

    PubMed

    Lee, Kang-Kon; Yum, Keun-Sang

    2012-09-01

    Endotoxins are known to be associated with the occurrence of various chronic diseases. This study was conducted to investigate the role of endotoxins in the pathogenesis of colon polyps through a case-control study. A total of 145 subjects (74 subjects in the polyp group and 71 subjects in the control group) had undergone a colonoscopy. Age, body mass index (BMI) and endotoxin levels were found to be significantly higher in the polyp group than in the control group. The endotoxin level was still significantly higher in the polyp group than in the control group, even after age and BMI had been adjusted (polyp group 0.108 ± 0.007 EU/mL, control group 0.049 ± 0.008 EU/mL, P < 0.001). In subgroup analysis, the endotoxin level significantly increased in accordance with the number of colon polyps (one-polyp group, 0.088 ± 0.059 EU/mL; two-polyp group, 0.097 ± 0.071 EU/mL; three-or-more-polyp group, 0.149 ± 0.223 EU/mL). The endotoxin levels also significantly increased in groups with tubular adenoma with high-grade dysplasia (hyperplastic polyp group, 0.109 ± 0.121 EU/mL; tubular adenoma with low grade dysplasia group, 0.103 ± 0.059 EU/mL; tubular adenoma with high grade dysplasia group, 2.915 ± 0.072 EU/mL). In conclusion, the serum level of endotoxins is quantitatively correlated with colon polyps.

  13. Effects of Puerariae Radix Extract on Endotoxin Receptors and TNF-α Expression Induced by Gut-Derived Endotoxin in Chronic Alcoholic Liver Injury.

    PubMed

    Peng, Jing-Hua; Cui, Tuan; Sun, Zhao-Lin; Huang, Fu; Chen, Liang; Xu, Lin; Feng, Qin; Hu, Yi-Yang

    2012-01-01

    Kudzu (Pueraria lobata) is one of the earliest medicinal plants used to treat alcohol abuse in traditional Chinese medicine for more than a millennium. However, little is known about its effects on chronic alcoholic liver injury. Therefore, the present study observed the effects of puerariae radix extract (RPE) on chronic alcoholic liver injury as well as Kupffer cells (KCs) activation to release tumor necrosis factor alpha (TNF-α) induced by gut-derived endotoxin in rats and macrophage cell line. RPE was observed to alleviate the pathological changes and lipids deposition in liver tissues as well as the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic gamma-glutamyl transpeptidase (GGT) activity. Meanwhile, RPE inhibited KCs activation and subsequent hepatic TNF-α expression and downregulated the protein expression of endotoxin receptors, lipopolysaccharide binding protein (LBP), CD14, Toll-like receptor (TLR) 2, and TLR4 in chronic alcohol intake rats. Furthermore, an in vitro study showed that RPE inhibited the expression of TNF-α and endotoxin receptors, CD14 and TLR4, induced by LPS in RAW264.7 cells. In summary, this study demonstrated that RPE mitigated liver damage and lipid deposition induced by chronic alcohol intake in rats, as well as TNF-α release, protein expression of endotoxin receptors in vivo or in vitro. PMID:23133491

  14. Delta II commercial space transportation

    NASA Astrophysics Data System (ADS)

    Meyers, J. F.

    1988-07-01

    Delta II is an upgraded variant of the Delta family of launch vehicles that has been in use by NASA since 1960. Among the design improvements incorporated by Delta II is a cryogenic-propellant second stage, a 2.89-m diameter satellite-protecting nose fairing, graphite/epoxy solid rocket motor cases, and 12:1 main engine expansion nozzle. The manufacturer/operator offers Delta II customers a dedicated, single satellite launch capability fully tailored to the given spacecraft's unique mission requirements.

  15. Characterization of endotoxin and 3-hydroxy fatty acid levels in air and settled dust from commercial aircraft cabins.

    PubMed

    Hines, C J; Waters, M A; Larsson, L; Petersen, M R; Saraf, A; Milton, D K

    2003-06-01

    Endotoxin was measured in air and dust samples collected during four commercial aircraft flights. Samples were analyzed for endotoxin biological activity using the Limulus assay. 3-hydroxy fatty acids (3-OH FA) of carbon chain lengths C10:0-C18:0 were determined in dust by gas chromatography-ion trap tandem mass spectrometry. The geometric mean (geometric standard deviation) endotoxin air level was 1.5 EU/m3 (1.9, n = 28); however, significant differences were found by flight within aircraft type. Mean endotoxin levels were significantly higher in carpet dust than in seat dust (140 +/- 81 vs. 51 +/- 25 EU/mg dust, n = 32 each, P < 0.001). Airborne endotoxin levels were not significantly related to either carpet or seat dust endotoxin levels. Mean 3-OH FA levels were significantly higher in carpet dust than in seat dust for C10:2, C12:0, and C14:0 (P < 0.001 for each), while the mean level of C16:0 was significantly higher in seat dust than in carpet dust (P < 0.01). Carpet dust endotoxin was significantly, but moderately, correlated with 3-OH-C12:0 and 3-OH-C14:0 (Pearson r = 0.52 and 0.48, respectively), while correlation of seat dust endotoxin with individual 3-OH FAs depended on the test statistic used. Mean endotoxin potency was significantly higher for carpet dust than for seat dust (6.3 +/- 3.0 vs. 3.0 +/- 1.4 EU/pmol LPS, P < 0.0001). Mean endotoxin levels in the air and dust of commercial aircraft cabins were generally higher than mean levels reported in homes and office buildings. These results suggest that exposure route and dust source are important considerations when relating endotoxin exposure to specific health outcomes.

  16. Meteo-climatic conditions influence the contribution of endotoxins to PM10 in an urban polluted environment.

    PubMed

    Traversi, D; Alessandria, L; Schilirò, T; Chiadò Piat, S; Gilli, G

    2010-02-01

    A decrease in inhalable particulate matter (PM10) pollution is a top priority in urban areas of northern Italy. The sources of PM10 are both anthropogenic and natural. The former have been broadly investigated while the latter are less well known. Endotoxins are natural compounds of PM10 and are potentially toxic. Endotoxins are part of the outer membrane of Gram-negative bacteria. Their health effects are linked to environmental exposure. The effects mainly consist of respiratory symptoms, including pulmonary function decline. The occurrence of endotoxins has been proven in several occupational environments where organic materials supply an optimal substrate for bacteria growth. Knowledge about the presence of these contaminants in the environment is limited. The aim of this work is to evaluate the endotoxin levels of PM10 in the urban air of Turin, and to investigate the influence of seasonal and meteo-climatic factors. The sampling was conducted from January to December 2007. Endotoxin determination was performed by an LAL assay after extraction optimization. The PM10 levels ranged from 11.90 to 104.74 microg/m(3) (48.28 +/- 23.09) while the endotoxin levels ranged between 0.09 and 0.94 EU/m(3) (0.42 +/- 0.23). The seasonal trends of PM10 and endotoxin are inversely proportional. There is a statistically significant correlation between endotoxin and temperature (r = 0.532 p < 0.01), as well as between endotoxin and relative humidity (r = -0.457 p < 0.01). However, temperature has a predominant role. We observed that urban endotoxin concentrations are narrow in range and that the contribution of endotoxins to the total PM10 is only two millionths.

  17. Shanghai Delta Complex

    SciTech Connect

    Hart, R.E.; Hoffman, P.F.; Parker, R.W.

    1988-01-01

    The upper Eocene Yegua Formation expands dramatically across a regional system of growth faults into an area generally 12-15 km wide, extending at least from the western edge of the Houston sale dome basin to the San Marcos arch. Within this area, the expanded Yegua trend has yielded, since 1982, at least seven noteworthy discoveries: Toro Grande and Lost Bridge fields in Jackson County, and Black Owl, Shanghai, Shanghai East, El Campo, and Phase Four fields in Wharton County. During each of several postulated Yegua sea level drops, this flexure became a focal point for deltaic deposition of excellent reservoir-quality sands. Shanghai, Shanghai East, and El Campo fields are located within what the writers have labeled the ''Shanghai delta complex.'' Integration of seismic and well data in this vicinity shows a marked increase in the expansion indices of growth faults, and moderately thick progradational sand sequences have accumulated immediately downthrow. This structural-stratigraphic pattern, as well as internal bedding characteristics and other lithologic data observed, is believed typical of deltas deposited along the Yegua shelf margin.

  18. Holden Crater Delta

    NASA Technical Reports Server (NTRS)

    2006-01-01

    [figure removed for brevity, see original site] Context image for PIA03694 Holden Crater Delta

    This fan-shaped delta deposit is located in Holden Crater.

    Image information: VIS instrument. Latitude -27.3N, Longitude 324.5E. 17 meter/pixel resolution.

    Note: this THEMIS visual image has not been radiometrically nor geometrically calibrated for this preliminary release. An empirical correction has been performed to remove instrumental effects. A linear shift has been applied in the cross-track and down-track direction to approximate spacecraft and planetary motion. Fully calibrated and geometrically projected images will be released through the Planetary Data System in accordance with Project policies at a later time.

    NASA's Jet Propulsion Laboratory manages the 2001 Mars Odyssey mission for NASA's Office of Space Science, Washington, D.C. The Thermal Emission Imaging System (THEMIS) was developed by Arizona State University, Tempe, in collaboration with Raytheon Santa Barbara Remote Sensing. The THEMIS investigation is led by Dr. Philip Christensen at Arizona State University. Lockheed Martin Astronautics, Denver, is the prime contractor for the Odyssey project, and developed and built the orbiter. Mission operations are conducted jointly from Lockheed Martin and from JPL, a division of the California Institute of Technology in Pasadena.

  19. Determination of endotoxin through an aptamer-based impedance biosensor.

    PubMed

    Su, Wenqiong; Lin, Meng; Lee, Hyuck; Cho, MiSuk; Choe, Woo-Seok; Lee, Youngkwan

    2012-02-15

    Lipopolysaccharide (LPS) often referred to endotoxin is an undesirable impurity frequently entrained with various recombinant protein therapeutics and plasmid DNA (pDNA) vaccines of bacterial origin. The inherent toxicities (e.g. fever, hypotension, shock and death) of LPS render its early and sensitive detection essential for several biological assays and/or parenteral administrations of biotherapeutics. In this study, an electrochemical biosensor using an LPS specific single stranded DNA (ssDNA) aptamer as a probe was developed. Amine-terminated aptamer exhibiting high affinity (K(d)=11.9 nM) to LPS was immobilized on a gold electrode using 3-mercaptopropionic acid (MPA) as a linker. Each step of the modification process was characterized by cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS). A good linear relationship of the changes in the charge-transfer resistance (ΔR(et)) and the logarithmic value of LPS concentration was demonstrated in a broad dynamic detection range of 0.001-1 ng/ml. Furthermore, the aptasensor showed a high selectivity to LPS despite the presence of pDNA, RNA and bovine serum albumin (BSA) and could be regenerated in low pH condition, offering a promising option for detecting LPS often present in a complex milieu.

  20. Air-sampled Filter Analysis for Endotoxins and DNA Content.

    PubMed

    Lang-Yona, Naama; Mazar, Yinon; Pardo, Michal; Rudich, Yinon

    2016-01-01

    Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods. PMID:27023725

  1. Regional blood flow during continuous low-dose endotoxin infusion

    SciTech Connect

    Fish, R.E.; Lang, C.H.; Spitzer, J.A.

    1986-01-01

    Escherichia coli endotoxin (ET) was administered to adult rats by continuous IV infusion from a subcutaneously implanted osmotic pump (Alzet). Cardiac output and regional blood flow were determined by the radiolabeled microsphere method after 6 and 30 hr of ET or saline infusion. Cardiac output (CO) of ET rats was not different from time-matched controls, whereas arterial pressure was 13% lower after 30 hr of infusion. After both 6 and 30 hr of ET, pancreatic blood flow and percentage of cardiac output were lower than in controls. Estimated portal venous flow was decreased at each time point, and an increased hepatic arterial flow (significant after 30 hr) resulted in an unchanged total hepatic blood flow. Blood flow to most other tissues, including epididymal fat, muscle, kidneys, adrenals, and gastrointestinal tract, was similar between treatments. Maintenance of blood flow to metabolically important tissues indicates that the previously reported alterations in in vitro cellular metabolism are not due to tissue hypoperfusion. Earlier observations of in vitro myocardial dysfunction, coexistent with the significant impairment in pancreatic flow, raise the possibility that release of a myocardial depressant factor occurs not only in profound shock but also under less severe conditions of sepsis and endotoxemia.

  2. CCL2 mediates the circadian response to low dose endotoxin.

    PubMed

    Duhart, José M; Brocardo, Lucila; Mul Fedele, Malena L; Guglielmotti, Angelo; Golombek, Diego A

    2016-09-01

    The mammalian circadian system is mainly originated in a master oscillator located in the suprachiasmatic nuclei (SCN) in the hypothalamus. Previous reports from our and other groups have shown that the SCN are sensitive to systemic immune activation during the early night, through a mechanism that relies on the action of proinflammatory factors within this structure. Chemokine (C-C motif) ligand 2 (CCL2) is induced in the brain upon peripheral immune activation, and it has been shown to modulate neuronal physiology. In the present work we tested whether CCL2 might be involved in the response of the circadian clock to peripheral endotoxin administration. The CCL2 receptor, C-C chemokine receptor type 2 (CCR2), was detected in the SCN of mice, with higher levels of expression during the early night, when the clock is sensitive to immune activation. Ccl2 was induced in the SCN upon intraperitoneal lipopolysaccharide (LPS) administration. Furthermore, mice receiving an intracerebroventricular (Icv) administration of a CCL2 synthesis inhibitor (Bindarit), showed a reduction LPS-induced circadian phase changes and Icv delivery of CCL2 led to phase delays in the circadian clock. In addition, we tested the possibility that CCL2 might also be involved in the photic regulation of the clock. Icv administration of Bindarit did not modify the effects of light pulses on the circadian clock. In summary, we found that CCL2, acting at the SCN level is important for the circadian effects of immune activation.

  3. Determination of endotoxin through an aptamer-based impedance biosensor.

    PubMed

    Su, Wenqiong; Lin, Meng; Lee, Hyuck; Cho, MiSuk; Choe, Woo-Seok; Lee, Youngkwan

    2012-02-15

    Lipopolysaccharide (LPS) often referred to endotoxin is an undesirable impurity frequently entrained with various recombinant protein therapeutics and plasmid DNA (pDNA) vaccines of bacterial origin. The inherent toxicities (e.g. fever, hypotension, shock and death) of LPS render its early and sensitive detection essential for several biological assays and/or parenteral administrations of biotherapeutics. In this study, an electrochemical biosensor using an LPS specific single stranded DNA (ssDNA) aptamer as a probe was developed. Amine-terminated aptamer exhibiting high affinity (K(d)=11.9 nM) to LPS was immobilized on a gold electrode using 3-mercaptopropionic acid (MPA) as a linker. Each step of the modification process was characterized by cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS). A good linear relationship of the changes in the charge-transfer resistance (ΔR(et)) and the logarithmic value of LPS concentration was demonstrated in a broad dynamic detection range of 0.001-1 ng/ml. Furthermore, the aptasensor showed a high selectivity to LPS despite the presence of pDNA, RNA and bovine serum albumin (BSA) and could be regenerated in low pH condition, offering a promising option for detecting LPS often present in a complex milieu. PMID:22182428

  4. Systemic response to low-dose endotoxin infusion in cats.

    PubMed

    DeClue, Amy E; Williams, Kurt J; Sharp, Claire; Haak, Carol; Lechner, Elizabeth; Reinero, Carol R

    2009-12-15

    Sepsis is a common problem in feline patients and is associated with substantial morbidity and mortality. There has been little research investigating the physiologic response to bacterial infection in cats, in part because appropriate models have not been developed. The objective of this study was to characterize the response to low-dose LPS infusion in conscious, healthy cats. Measures of systemic inflammation, hemodynamic stability, coagulation, metabolic function, and organ damage were compared between placebo and low-dose LPS infusion (2mcg/kg/hx4h, IV) in cats, with each cat serving as its own control. Markers of systemic inflammation including temperature, plasma TNF activity, IL-6, CXCL-8 and IL-10 concentrations were significantly increased and white blood cell counts were significantly decreased after LPS infusion. A biphasic hypotensive response was observed after initiation of LPS infusion without concurrent tachycardia. Additionally, LPS administration significantly increased blood glucose, lactate and creatinine concentrations. Patchy alveolar congestion, multifocal acute alveolar epithelial necrosis, and mild pulmonary edema were noted in the lungs along with acute centrilobular hepatocellular necrosis, and mild lymphocyte apoptosis in the spleen and/or intestinal Peyer's patches. No biologically significant alterations in coagulation parameters developed after LPS infusion. Low-dose LPS infusion in cats induced systemic inflammation, hemodynamic derangement, metabolic alterations and mild organ damage. Low-dose endotoxin infusion is a viable pre-clinical model to study naturally developing sepsis in cats.

  5. Hemodynamic effects of isoproterenol in canine endotoxin shock

    PubMed Central

    Starzecki, Boleslaw; Spink, Wesley W.

    1968-01-01

    Myocardial function and peripheral hemodynamic alterations were measured through the late stages of canine endotoxin shock. 60 min postendotoxin paired animals were given infusions of either 5 ml/kg per hr of 5% dextrose or dextrose plus isoproterenol (0.25 μg/kg per min). Comparable blood lactic and pyruvic acid levels were determined, the excess lactic acid calculated, and pH values were obtained. During the initial stages the classic pattern of hemodynamic alterations was observed; an excess of lactic acid appeared and the pH decreased. Outstanding was evidence of markedly reduced myocardial function in the late stages of shock with progressive rise in left ventricular end diastolic pressure (LVEDP), low cardiac index, rise of central venous pressure, increased central blood volume, tachycardia, and declining arterial pressure. Analyses of left ventricular function curves also indicated myocardial failure. Infusion of dextrose alone failed to decrease mortality rate (10 of 18 dying), whereas the rate was significantly decreased with isoproterenol (2 of 18). Dextrose infusion did not benefit myocardial function. Isoproterenol resulted in a marked improvement in myocardial action with a significant increase in heart work associated with, yet very minor, increments of LVEDP. In addition, tachycardia subsided, peripheral resistance decreased, and the blood pressure stabilized. The prognostic value of excess lactic acid was doubtful but a progressive fall in later stages was associated with survival. Images PMID:5676517

  6. Translocated LPS Might Cause Endotoxin Tolerance in Circulating Monocytes of Cystic Fibrosis Patients

    PubMed Central

    del Fresno, Carlos; Alenda, Raquel; Gómez-Piña, Vanesa; Fernández-Ruíz, Irene; Siliceo, María; Jurado, Teresa; Toledano, Victor; Arnalich, Francisco; García-Río, Francisco; López-Collazo, Eduardo

    2011-01-01

    Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes. PMID:22216320

  7. Toll-like receptor and tumour necrosis factor dependent endotoxin-induced acute lung injury

    PubMed Central

    Togbe, Dieudonnée; Schnyder-Candrian, Silvia; Schnyder, Bruno; Doz, Emilie; Noulin, Nicolas; Janot, Laure; Secher, Thomas; Gasse, Pamela; Lima, Carla; Coelho, Fernando Rodrigues; Vasseur, Virginie; Erard, François; Ryffel, Bernhard; Couillin, Isabelle; Moser, Rene

    2007-01-01

    Recent studies on endotoxin/lipopolysaccharide (LPS)-induced acute inflammatory response in the lung are reviewed. The acute airway inflammatory response to inhaled endotoxin is mediated through Toll-like receptor 4 (TLR4) and CD14 signalling as mice deficient for TLR4 or CD14 are unresponsive to endotoxin. Acute bronchoconstriction, tumour necrosis factor (TNF), interleukin (IL)-12 and keratinocyte-derived chemokine (KC) production, protein leak and neutrophil recruitment in the lung are abrogated in mice deficient for the adaptor molecules myeloid differentiation factor 88 (MyD88) and Toll/Interleukin-1 receptor (TIR)-domain-containing adaptor protein (TIRAP), but independent of TIR-domain-containing adaptor-inducing interferon-beta (TRIF). In particular, LPS-induced TNF is required for bronchoconstriction, but dispensable for inflammatory cell recruitment. Lipopolysaccharide induces activation of the p38 mitogen-activated protein kinase (MAPK). Inhibition of pulmonary MAPK activity abrogates LPS-induced TNF production, bronchoconstriction, neutrophil recruitment into the lungs and broncho-alveolar space. In conclusion, TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin are dependent on TLR4/CD14/MD2 expression using the adapter proteins TIRAP and MyD88, while TRIF, IL-1R1 or IL-18R signalling pathways are dispensable. Further downstream in this axis of signalling, TNF blockade reduces only acute bronchoconstriction, while MAPK inhibition abrogates completely endotoxin-induced inflammation. PMID:18039275

  8. Effect of deployment time on endotoxin and allergen exposure assessment using electrostatic dust collectors.

    PubMed

    Kilburg-Basnyat, Brita; Metwali, Nervana; Thorne, Peter S

    2015-01-01

    The electrostatic dust collector (EDC) is a passive dust sampling device for exposure assessment of airborne endotoxin and possibly allergens. EDCs consist of a non-conducting plastic folder holding two or four electrostatic cloths of defined area. The sampling time needed to achieve detectable and reproducible loading for bioaerosols has not been systematically evaluated. Thus, in 15 Iowa farm homes EDCs were deployed for 7-, 14-, and 28-day sampling periods to determine if endotoxin and allergens could be quantified and if loading rates were uniform over time, i.e. if loads doubled from 7 to 14 days or 14 to 28 days and quadrupled from 7 to 28 days. Loadings between left and right paired EDC cloths were not significantly different and were highly correlated for endotoxin, total protein, and cat (Fel d1), dog (Can f1), and mouse (Mus m1) allergens (P < 0.001). EDCs performed especially well for endotoxin sampling with close agreement between paired samples (Pearson r = 0.96, P < 0.001). Endotoxin loading of the EDCs doubled from 7- to 14-day deployments as hypothesized although the loading rate decreased from 14 to 28 days of sampling with only a 1.38-fold increase. Allergen exposure assessment using EDCs was overall less satisfactory. Although there was reasonable agreement between paired samples, only exposures to cat, dog, and mouse allergens were reliable and these only at the longer deployment times.

  9. Detergent inhibits 70-90% of responses to intravenous endotoxin in awake sheep.

    PubMed

    Staub, N C; Longworth, K E; Serikov, V; Jerome, E H; Elsasser, T

    2001-05-01

    Sheep have reactive pulmonary intravascular macrophages, which are essential for the marked pulmonary vascular response to infusions of small quantities of endotoxin. In another species with reactive pulmonary intravascular macrophages, horses, our laboratory found that an intravenous biosafe detergent, tyloxapol, inhibited some systemic and pulmonary responses to endotoxin (Longworth KE, Smith BL, Staub NC, Steffey EP, and Serikov V. Am J Vet Res 57: 1063-1066, 1996). We determined whether the same detergent would inhibit endotoxin responses in awake sheep. In 10 awake, instrumented sheep with chronic lung lymph fistulas, we did a control experiment by intravenously infusing 1 microg/kg Escherichia coli endotoxin. One week later, we gave 40 micromol/kg tyloxapol intravenously 1-4 h before infusing the same dose of endotoxin. In these paired studies, we compared pulmonary hemodynamics, lung lymph dynamics, body temperature, circulating leukocyte concentrations, and circulating tumor necrosis factor for 6 h. In all 10 sheep, tyloxapol blocked 80-90% of the pulmonary responses and 70-90% of the systemic responses. Tyloxapol is safe, inexpensive, easy to use, and effective immediately. It may be a clinically useful approach to contravening many of the effects of endotoxemia, in humans as well as animals.

  10. Endotoxin concentration in poultry houses for laying hens kept i