Search results for "binding"
showing 10 items of 3896 documents
Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentida…
2014
ABSTRACT Bacillus thuringiensis Cry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genus Cylas , which are important pests of sweet potato worldwide and particularly in sub-Saharan Africa. However, relatively little is known about the processing and binding interactions of these coleopteran-specific Cry proteins. The aim of the present study was to determine whether Cry3Bb, Cry3Ca, and Cry7Aa proteins have shared binding sites in Cylas puncticollis to orient the pest resistance strategy by genetic transformation. Interestingly, processing of the 129-kDa Cry7Aa protoxin using commercial trypsin or chymotrypsin rendered two fragments of about 70 kDa and 65 kDa. N-…
Lyophilization of lepidopteran midguts: a preserving method for Bacillus thuringiensis toxin binding studies
2004
Binding assays with brush border membrane vesicles (BBMV) from insect midguts are commonly used in the study of the interactions between Bacillus thuringiensis Cry toxins and their receptors. Collaboration between laboratories often require that frozen insect samples are sent in dry ice. Because of customs restrictions and delays, sample thawing is always a risk and often the biological material becomes ruined during shipping. We have tested lyophilization as an alternative method for preserving insect midguts for binding studies with B. thuringiensis Cry toxins. For this purpose, BBMV were prepared from both frozen and lyophilized midguts from three lepidopteran species: Spodoptera exigua,…
Selective inhibition of binding of Bacillus thuringiensis Cry1Ab toxin to cadherin-like and aminopeptidase proteins in brush-border membranes and dis…
2007
Binding analyses with denatured epithelial membrane proteins from Bt (Bacillus thuringiensis) demonstrated at least two kinds of proteins, APNs (aminopeptidases N) and cadherin-like proteins, as possible receptors for the Cry1A class of Bt toxins. Two alternative models have been proposed, both based on initial toxin binding to a cadherin-like protein, but one involving APN and the other not. We have used two Bombyx mori strains (J65 and Kin), which are highly susceptible to Cry1Ab, to study the role of these two types of receptors on Cry1Ab toxin binding and cytotoxicity by means of the inhibitory effect of antibodies. BBMVs (brush-border membrane vesicles) of strain J65 incubated with lab…
Binding of Bacillus thuringiensis toxins in resistant and susceptible strains of pink bollworm (Pectinophora gossypiella)
2003
Abstract Evolution of resistance by pests could cut short the success of transgenic plants producing toxins from Bacillus thuringiensis, such as Bt cotton. The most common mechanism of insect resistance to B. thuringiensis is reduced binding of toxins to target sites in the brush border membrane of the larval midgut. We compared toxin binding in resistant and susceptible strains of Pectinophora gossypiella, a major pest of cotton worldwide. Using Cry1Ab and Cry1Ac labeled with 125I and brush border membrane vesicles (BBMV), competition experiments were performed with unlabeled Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ca, Cry1Ja, Cry2Aa, and Cry9Ca. In the susceptible strain, Cry1Aa, Cry1Ab, Cry1…
Bacillus thuringiensis crystal proteins CRY1Ab and CRY1Fa share a high affinity binding site in Plutella xylostella (L.).
1996
The future success of Bacillus thuringiensis based insecticides depends in part on our ability to prevent insects from developing resistance against their insecticidal crystal proteins. Two recent papers indicated cross-resistance between Cry1A proteins and Cry1Fa in two different insect species (Tabashnik et al., 1994, Appl. Environ. Microbiol. 60, 4627-4629; Gould et al., 1995, J. Econ. Entomol. 88, 1545-1559). Brush border membrane vesicles were prepared from Plutella xylostella and used in binding assays with 125I-labeled trypsin-activated crystal proteins. Competition experiments showed that Cry1Fa competed with Cry1Ab for a same binding site, though the latter still bound to a differe…
Development and Characterization of Diamondback Moth Resistance to Transgenic Broccoli Expressing High Levels of Cry1C
2000
ABSTRACT A field-collected colony of the diamondback moth, Plutella xylostella , had 31-fold resistance to Cry1C protoxin of Bacillus thuringiensis . After 24 generations of selection with Cry1C protoxin and transgenic broccoli expressing a Cry1C protein, the resistance that developed was high enough that neonates of the resistant strain could complete their entire life cycle on transgenic broccoli expressing high levels of Cry1C. After 26 generations of selection, the resistance ratios of this strain to Cry1C protoxin were 12,400- and 63,100-fold, respectively, for the neonates and second instars by a leaf dip assay. The resistance remained stable until generation 38 (G38) under continuous…
Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen.
2012
ABSTRACT 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 differe…
In Vivo and In Vitro Binding of Vip3Aa to Spodoptera frugiperda Midgut and Characterization of Binding Sites by 125 I Radiolabeling
2014
ABSTRACT Bacillus thuringiensis vegetative insecticidal proteins (Vip3A) have been recently introduced in important crops as a strategy to delay the emerging resistance to the existing Cry toxins. The mode of action of Vip3A proteins has been studied in Spodoptera frugiperda with the aim of characterizing their binding to the insect midgut. Immunofluorescence histological localization of Vip3Aa in the midgut of intoxicated larvae showed that Vip3Aa bound to the brush border membrane along the entire apical surface. The presence of fluorescence in the cytoplasm of epithelial cells seems to suggest internalization of Vip3Aa or a fragment of it. Successful radiolabeling and optimization of the…
Cyclic AMP-mediated upregulation of the expression of neuronal NO synthase in human A673 neuroepithelioma cells results in a decrease in the level of…
2004
The expression level of neuronal nitric oxide synthase (nNOS) can vary depending on the (patho)physiological conditions. Here we document a marked induction of nNOS mRNA, protein, and total NO production in response to dibutyryl cyclic AMP (db-cAMP) in human A673 neuroepithelial cells. However, the upregulation of nNOS was associated with a decreased level of production of bioactive NO and by an increase in the level of generation of reactive oxygen species (ROS). ROS production could be prevented by the NOS inhibitor L-NAME, suggesting nNOS itself is involved in ROS generation. Sepiapterin supplementation of db-cAMP-treated A673 cells could restore full bioactive NO production, most likely…
Gene Expression Analyses during Spontaneous Reversal of Cardiomyopathy in Mice with Repressed Nuclear CUG-BP, Elav-Like Family (CELF) Activity in Hea…
2015
CUG-BP, Elav-like family (CELF) proteins regulate cell type- and developmental stage-specific alternative splicing in the heart. Repression of CELF-mediated splicing activity via expression of a nuclear dominant negative CELF protein in heart muscle was previously shown to induce dysregulation of alternative splicing, cardiac dysfunction, cardiac hypertrophy, and dilated cardiomyopathy in MHC-CELFΔ transgenic mice. A “mild” line of MHC-CELFΔ mice that expresses a lower level of the dominant negative protein exhibits cardiac dysfunction and myopathy at a young age, but spontaneously recovers normal cardiac function and heart size with age despite the persistence of splicing defects. To the b…