scholarly journals A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

2000 ◽  
Vol 66 (4) ◽  
pp. 1553-1558 ◽  
Author(s):  
Carolina Rausell ◽  
Amparo Consuelo Martínez-Ramírez ◽  
Inmaculada García-Robles ◽  
María Dolores Real
Keyword(s):  
Bacillus Thuringiensis ◽  
Larval Development ◽  
Binding Sites ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Thaumetopoea Pityocampa ◽  
Binding Assays ◽  
Forest Pests ◽  
First Instar ◽  
Nun Moth

ABSTRACT The insecticidal activity and receptor binding properties ofBacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) andLymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with 125I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age.

scholarly journals Interaction of Bacillus thuringiensis Cry1 and Vip3A Proteins with Spodoptera frugiperda Midgut Binding Sites

2009 ◽  
Vol 75 (7) ◽  
pp. 2236-2237 ◽  
Author(s):  
Janete A. D. Sena ◽  
Carmen Sara Hernández-Rodríguez ◽  
Juan Ferré
Keyword(s):  
Bacillus Thuringiensis ◽  
Spodoptera Frugiperda ◽  
Binding Sites ◽  
Specific Binding ◽  
Binding Assays ◽  
Binding Interactions ◽  
Specific Binding Sites

ABSTRACT Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

scholarly journals Specific Binding of Bacillus thuringiensis Cry2A Insecticidal Proteins to a Common Site in the Midgut of Helicoverpa Species

2008 ◽  
Vol 74 (24) ◽  
pp. 7654-7659 ◽  
Author(s):  
Carmen Sara Hernández-Rodríguez ◽  
Adri Van Vliet ◽  
Nadine Bautsoens ◽  
Jeroen Van Rie ◽  
Juan Ferré
Keyword(s):  
Binding Sites ◽  
Mode Of Action ◽  
Helicoverpa Zea ◽  
Tertiary Structure ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Binding Assays ◽  
Cry Toxins ◽  
Long Time ◽  
Competition Assays

ABSTRACT For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of K d (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding R t (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins.

scholarly journals Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Jian Jiang ◽  
Ying Huang ◽  
Changlong Shu ◽  
Mario Soberón ◽  
Alejandra Bravo ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Sites ◽  
Binding Proteins ◽  
Insect Pest ◽  
Membrane Vesicles ◽  
Economic Losses ◽  
Binding Assays ◽  
Uncharacterized Protein ◽  
Content Type ◽  
Lepidopteran Insect

ABSTRACT The Bacillus thuringiensis strain HBF-18 (CGMCC 2070), containing two cry genes (cry8-like and cry8Ga), is toxic to Holotrichia oblita larvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) in H. oblita larvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs from H. oblita, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut from H. oblita larvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the H. oblita midgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with the H. oblita transcriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 of Tribolium castaneum, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin. IMPORTANCE Holotrichia oblita is one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses. H. oblita is difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins from Bacillus thuringiensis were shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control of H. oblita and for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins against H. oblita larvae.

scholarly journals Binding Sites for Bacillus thuringiensis Cry2Ae Toxin on Heliothine Brush Border Membrane Vesicles Are Not Shared with Cry1A, Cry1F, or Vip3A Toxin

2011 ◽  
Vol 77 (10) ◽  
pp. 3182-3188 ◽  
Author(s):  
C. Gouffon ◽  
A. Van Vliet ◽  
J. Van Rie ◽  
S. Jansens ◽  
J. L. Jurat-Fuentes
Keyword(s):  
Bacillus Thuringiensis ◽  
Brush Border ◽  
Binding Sites ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Target Range ◽  
Content Type ◽  
Bt Toxins

ABSTRACTThe use of combinations ofBacillus thuringiensis(Bt) toxins with diverse modes of action for insect pest control has been proposed as the most efficient strategy to increase target range and delay the onset of insect resistance. Considering that most cases of cross-resistance to Bt toxins in laboratory-selected insect colonies are due to alteration of common toxin binding sites, independent modes of action can be defined as toxins sharing limited or no binding sites in brush border membrane vesicles (BBMV) prepared from the target insect larvae. In this paper, we report on the specific binding of Cry2Ae toxin to binding sites on BBMV from larvae of the three most commercially relevant heliothine species,Heliothis virescens,Helicoverpa zea, andHelicoverpa armigera. Using chromatographic purification under reducing conditions before labeling, we detected specific binding of radiolabeled Cry2Ae, which allowed us to perform competition assays using Cry1Ab, Cry1Ac, Cry1Fa, Vip3A, Cry2Ae, and Cry2Ab toxins as competitors. In these assays, Cry2Ae binding sites were shared with Cry2Ab but not with the tested Cry1 or Vip3A toxins. Our data support the use of Cry2Ae toxin in combination with Cry1 or Vip3A toxins in strategies to increase target range and delay the onset of heliothine resistance.

scholarly journals Shared Binding Sites for the Bacillus thuringiensis Proteins Cry3Bb, Cry3Ca, and Cry7Aa in the African Sweet Potato Pest Cylas puncticollis (Brentidae)

2014 ◽  
Vol 80 (24) ◽  
pp. 7545-7550 ◽  
Author(s):  
Patricia Hernández-Martínez ◽  
Natalia Mara Vera-Velasco ◽  
María Martínez-Solís ◽  
Marc Ghislain ◽  
Juan Ferré ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Sweet Potato ◽  
Binding Sites ◽  
Specific Binding ◽  
Pest Resistance ◽  
Membrane Vesicles ◽  
Sub Saharan Africa ◽  
Receptor Binding Site ◽  
Content Type ◽  
Cylas Puncticollis

ABSTRACTBacillus thuringiensisCry3Bb, Cry3Ca, and Cry7Aa have been reported to be toxic against larvae of the genusCylas, 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 inCylas puncticollisto 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-terminal sequencing of the trypsin-activated Cry7Aa fragments revealed that processing occurs at Glu47for the 70-kDa form or Ile88for the 65-kDa form. Homologous binding assays showed specific binding of the two Cry3 proteins and the 65-kDa Cry7Aa fragment to brush border membrane vesicles (BBMV) fromC. puncticollislarvae. The 70-kDa fragment did not bind to BBMV. Heterologous-competition assays showed that Cry3Bb, Cry3Ca, and Cry7Aa (65-kDa fragment) competed for the same binding sites. Hence, our results suggest that pest resistance mediated by the alteration of a shared Cry receptor binding site might render all three Cry toxins ineffective.

Endothelin ETA and ETB receptors in postnatal intestine

2001 ◽  
Vol 280 (4) ◽  
pp. G555-G562 ◽  
Author(s):  
Craig A. Nankervis ◽  
David J. Dunaway ◽  
Charles E. Miller
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ischemia Reperfusion ◽  
Age Groups ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Site Model ◽  
Number Of Binding Sites ◽  
Competitive Binding Assays

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E− vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

EFFECT OF ENDOCRINE MANIPULATIONS ON GLUCOCORTICOID BINDING CAPACITY IN RAT SKELETAL MUSCLE

1978 ◽  
Vol 88 (1) ◽  
pp. 199-208 ◽  
Author(s):  
Michael Mayer ◽  
Fred Rosen
Keyword(s):  
Skeletal Muscle ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Female Rats ◽  
Receptor Protein ◽  
Slight Reduction ◽  
Binding Assays ◽  
Receptor Concentration ◽  
Streptozotocin Induced Diabetes

ABSTRACT [3H]Dexamethasone binding capacity in rat muscle cytosol was determined after various endocrine manipulations in an attempt to identify factors which might regulate the level of the cytoplasmic hormone receptor protein. Hypophysectomy and adrenalectomy markedly increased the specific binding of [3H]dexamethasone in skeletal muscle cytosol, while implantation of the MtT tumour which secretes ACTH and growth hormone, as well as treatment with glucocorticoids reduced the glucocorticoid specific binding. Since the effects of hypophysectomy and the MtT tumour depend on the presence of the adrenals, they appear to be mediated via changes in circulating glucocorticoid level. Alloxan- or streptozotocin-induced diabetes caused only a slight reduction in the binding of [3H]dexamethasone in muscle, suggesting that the enhanced responsiveness to glucocorticoids in diabetes is not due to increased glucocorticoid receptor activity. There is a sex-dependent effect on binding, female rats having a higher concentration of binding sites. Furthermore, treatment with the synthetic androgen fluoxymesterone or with glucocorticoids reduces binding, while oestradiol-17β enhances it. The changes in glucocorticoid binding capacity induced by the various endocrine manipulations appear to reflect mainly changes in receptor concentration rather than occupancy, since the binding assays were preformed after a suitable time allowance for removal of the administered hormones by metabolism.

scholarly journals Use of Bacillus thuringiensis Toxins for Control of the Cotton Pest Earias insulana (Boisd.) (Lepidoptera: Noctuidae)

2006 ◽  
Vol 72 (1) ◽  
pp. 437-442 ◽  
Author(s):  
María A. Ibargutxi ◽  
Anna Estela ◽  
Juan Ferré ◽  
Primitivo Caballero
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Sites ◽  
Specific Binding ◽  
Significant Inhibition ◽  
The Other ◽  
Cross Resistance ◽  
Earias Insulana ◽  
Cry Proteins ◽  
Cry Toxins ◽  
Laboratory Colony

ABSTRACT Thirteen of the most common lepidopteran-specific Cry proteins of Bacillus thuringiensis have been tested for their efficacy against newly hatched larvae of two populations of the spiny bollworm, Earias insulana. At a concentration of 100 μg of toxin per milliliter of artificial diet, six Cry toxins (Cry1Ca, Cry1Ea, Cry1Fa, Cry1Ja, Cry2Aa, and Cry2Ab) were not toxic at all. Cry1Aa, Cry1Ja, and Cry2Aa did not cause mortality but caused significant inhibition of growth. The other Cry toxins (Cry1Ab, Cry1Ac, Cry1Ba, Cry1Da, Cry1Ia, and Cry9Ca) were toxic to E. insulana larvae. The 50% lethal concentration values of these toxins ranged from 0.39 to 21.13 μg/ml (for Cry9Ca and Cry1Ia, respectively) for an E. insulana laboratory colony originating from Egypt and from 0.20 to 4.25 μg/ml (for Cry9Ca and Cry1Da, respectively) for a laboratory colony originating from Spain. The relative potencies of the toxins in the population from Egypt were highest for Cry9Ca and Cry1Ab, and they were both significantly more toxic than Cry1Ac and Cry1Ba, followed by Cry1Da and finally Cry1Ia. In the population from Spain, Cry9Ca was the most toxic, followed in decreasing order by Cry1Ac and Cry1Ba, and the least toxic was Cry1Da. Binding experiments were performed to test whether the toxic Cry proteins shared binding sites in this insect. 125I-labeled Cry1Ac and Cry1Ab and biotinylated Cry1Ba, Cry1Ia, and Cry9Ca showed specific binding to the brush border membrane vesicles from E. insulana. Competition binding experiments among these toxins showed that only Cry1Ab and Cry1Ac competed for the same binding sites, indicating a high possibility that this insect may develop cross-resistance to Cry1Ab upon exposure to Cry1Ac transgenic cotton but not to the other toxins tested.

The rainbow trout skeletal muscle β-adrenergic system: characterization and signaling

2003 ◽  
Vol 284 (3) ◽  
pp. R689-R697 ◽  
Author(s):  
Michel B. Lortie ◽  
Thomas W. Moon
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Specific Binding ◽  
Adrenergic System ◽  
Camp Production ◽  
Binding Assays ◽  
Single Class ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Adrenergic Antagonist ◽  
Number Of Binding Sites

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( K d in nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmax in fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1 · 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.

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