This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Thesis (Ph. D. in Biological Sciences)--Southern Methodist University. Includes bibliographical r... more Thesis (Ph. D. in Biological Sciences)--Southern Methodist University. Includes bibliographical references (leaves 89-99).
Biology curricula have been traditionally based on disciplinary approach to instructional topics ... more Biology curricula have been traditionally based on disciplinary approach to instructional topics like biochemistry, cell biology, immunology, microbiology, etc. However, biological sciences knowled...
A composition and method for killing fire ants is disclosed that includes the purified and activa... more A composition and method for killing fire ants is disclosed that includes the purified and activated Cry toxin from a novel strain of Bacillus thuringiensis, wherein the water soluble, partially purified, activated toxin is provided along with an attractant to kill the imported fire ant using a biodegradable, environmentally sound glycoprotein without a need for toxic chemicals.
A cDNA encoding a 200 kD receptor, BT-R2, from the pink boll worm, Pectinophora gossypiella, that... more A cDNA encoding a 200 kD receptor, BT-R2, from the pink boll worm, Pectinophora gossypiella, that binds specifically to a Bacillus thuringiensis toxin has been cloned, sequenced and characterized. The minimum toxin binding fragment has been identified. The BT-R2 cDNA permits the analysis of receptors in pink boll worm and other insects that affect crop growth and development, as well as, design assays for the cytotoxicity and binding affinity of potential pesticides. The clone and other methods described herein, permit the manipulation of natural and/or introduced homologous receptors and, thus, to specifically destroy organisms, tissues and/or cells of the target host.
The cadherin-related receptor of Manduca sexta, BTgiensis; Sf21 cells; protein expression; Cry1Ab... more The cadherin-related receptor of Manduca sexta, BTgiensis; Sf21 cells; protein expression; Cry1Ab; recom-R 1 , for the Cry1A family of Bacillus thuringiensis insecbinant soluble protein; cadherin-related. ticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector ␦Op-gp64. Recombinant BT-R 1 was released by The environmentally friendly insecticidal parasporal the Sf21 cells in soluble form into the culture medium crystal proteins produced by Bacillus thuringiensis (1) and represents approximately 58% of total BT-R 1 proare of great scientific interest because of their potency duced by the cells. The soluble protein was purified by and specificity to a wide range of insect pests (3, 27). The affinity chromatography using Cry1Ab toxin coupled specificity of B. thuringiensis toxins correlates directly to Sepharose 4B. The apparent molecular mass of puriwith the presence of high-affinity binding proteins presfied soluble recombinant BT-R 1 is 195 kDa. Radiolaent in the midgut of target insects (7, 26), supporting the beled toxin bound to purified soluble BT-R 1 with a K d hypothesis (25) that insect receptor molecules mediate value of 1.1 nM, which is similar to that of both memtoxicity of these toxins. brane-bound BT-R 1 in Sf21 cells and natural BT-R 1 from A high-affinity protein receptor, BT-R 1 , for the B. M. sexta larval midgut tissue. Binding of radiolabeled thuringiensis Cry1A family of lepidopteran-specific toxtoxin to soluble BT-R 1 was competitively inhibited by ins has been identified (17, 24) and cloned (25) from unlabeled Cry1Ab toxin but not by other Cry toxins Manduca sexta larval midgut epithelial cells (4, 9, 10). as was observed also for membrane-bound BT-R 1. The BT-R 1 is a cadherin-related protein with an apparent recombinant soluble protein was stable in culture molecular weight of 210 kDa (24). Expression of BT-R 1 medium for at least 3 days at 27؇C and for 7 days at 4؇C is tissue specific. Only epithelial cells of M. sexta midgut and exhibited toxin-binding properties similar to the tissue harbor BT-R 1 mRNA and its translated protein natural protein. Apparently, neither membrane associ-(Midboe and Bulla, unpublished results). The cDNA for ation nor the extent of glycosylation influences the BT-R 1 (GenBank Accession No. AF319973) encodes a binding affinity and specificity of BT-R 1. Approxiprotein (25) comprising four putative domains: ectodomately 1 mg of purified BT-R 1 was obtained per liter main (EC 3 ; amino acid residues 1-1464), membraneof insect cell culture supernatant, representing ϳ2 ؋ proximal extracellular domain (MPED; residues 1465-10 9 Sf21 cells. ᭧ 2001 Academic Press 1532), a single transmembrane domain (residues 1533-1557), and cytoplasmic domain (residues 1558-1717).
Proceedings of the National Academy of Sciences, 2006
Many pathogenic organisms and their toxins target host cell receptors, the consequence of which i... more Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg 2+ -dependent cytotoxic event upon binding of the toxin to its receptor BT-R 1 . Here we show that binding of Cry toxin to BT-R 1 provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G αs ) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifest...
The objective of this study was to explore the role of molecular oxidative damage and caloric int... more The objective of this study was to explore the role of molecular oxidative damage and caloric intake in the aging process. The concentration of 8-hydroxydeoxyguanosine (8-OHdG), a product of DNA oxidation, was compared in five different tissues of mice (skeletal muscle, brain, heart, liver and kidney) as a function of age and in response to dietary restriction. A comparison of 8-and 27-month-old mice indicated that the age-related increase in 8-OHdG concentration was greater in skeletal muscle, brain and heart, which are primarily composed of long-lived, post-mitotic cells, than in liver and kidney, which consist of slowdividing cells. Dietary restricted (DR) mice kept on 60% caloric intake as compared to the ad libitum-fed (AL) mice showed a lower concentration in 8-OHdG content in all the tissues compared to AL mice. The DR-related amelioration of DNA oxidative damage was greater in the post-mitotic tissues compared to those undergoing slow mitoses. Results support the hypothesis that oxidative damage to long-lived post-mitotic cells may be a key factor in the aging process.
Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increa... more Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound ෂ60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of 125 I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K d ෂ0.1 µM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.
Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal p... more Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R 1 (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K d ෂ 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R 1 revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R 1 cDNA, subsequently killing the cells. Recruitment of BT-R 1 by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2003
The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R (210-kDa), which cont... more The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R (210-kDa), which contains 12 1 ectodomain modules in association with a number of motifs potentially involved in interactions with cadherin and integrin. The molecule is a target receptor for Bacillus thuringiensis Cry1A toxins that bind to BT-R with high affinity 1 and specificity. BT-R is localized exclusively in the midgut epithelium. The amount of BT-R protein increases 1 1 dramatically during larval development, paralleling accumulation of its mRNA. The 59-UTR of the btr-1 gene contains sequence motifs that most likely recruit specific transcription factors, particularly, those that determine posterior patterning and that control intestinal cell proliferation, differentiation and identity during development. The increase in abundance of BT-R may be required to support not only the differentiation of the epithelial cells but also the establishment of 1 physiological function and structural integrity of the midgut during larval development in M. sexta. We believe that BT-R is essential to larval midgut epithelial organization during rapid cell proliferation and tissue growth in M. sexta 1 because disruption of such organization and functionality occasioned by the binding of the Cry1A toxins of B. thuringiensis to BT-R causes death to the insect.
The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringi... more The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxinreceptor interactions in cytotoxicity, we established a cellbased system utilizing High Five insect cells stably expressing BT-R 1 , the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R 1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R 1 , which apparently activates a Mg 2 þdependent cellular signaling pathway.
The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding ... more The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R 1 , a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Thesis (Ph. D. in Biological Sciences)--Southern Methodist University. Includes bibliographical r... more Thesis (Ph. D. in Biological Sciences)--Southern Methodist University. Includes bibliographical references (leaves 89-99).
Biology curricula have been traditionally based on disciplinary approach to instructional topics ... more Biology curricula have been traditionally based on disciplinary approach to instructional topics like biochemistry, cell biology, immunology, microbiology, etc. However, biological sciences knowled...
A composition and method for killing fire ants is disclosed that includes the purified and activa... more A composition and method for killing fire ants is disclosed that includes the purified and activated Cry toxin from a novel strain of Bacillus thuringiensis, wherein the water soluble, partially purified, activated toxin is provided along with an attractant to kill the imported fire ant using a biodegradable, environmentally sound glycoprotein without a need for toxic chemicals.
A cDNA encoding a 200 kD receptor, BT-R2, from the pink boll worm, Pectinophora gossypiella, that... more A cDNA encoding a 200 kD receptor, BT-R2, from the pink boll worm, Pectinophora gossypiella, that binds specifically to a Bacillus thuringiensis toxin has been cloned, sequenced and characterized. The minimum toxin binding fragment has been identified. The BT-R2 cDNA permits the analysis of receptors in pink boll worm and other insects that affect crop growth and development, as well as, design assays for the cytotoxicity and binding affinity of potential pesticides. The clone and other methods described herein, permit the manipulation of natural and/or introduced homologous receptors and, thus, to specifically destroy organisms, tissues and/or cells of the target host.
The cadherin-related receptor of Manduca sexta, BTgiensis; Sf21 cells; protein expression; Cry1Ab... more The cadherin-related receptor of Manduca sexta, BTgiensis; Sf21 cells; protein expression; Cry1Ab; recom-R 1 , for the Cry1A family of Bacillus thuringiensis insecbinant soluble protein; cadherin-related. ticidal toxins, was expressed in cultured Spodoptera frugiperda (Sf21) insect cells utilizing the expression vector ␦Op-gp64. Recombinant BT-R 1 was released by The environmentally friendly insecticidal parasporal the Sf21 cells in soluble form into the culture medium crystal proteins produced by Bacillus thuringiensis (1) and represents approximately 58% of total BT-R 1 proare of great scientific interest because of their potency duced by the cells. The soluble protein was purified by and specificity to a wide range of insect pests (3, 27). The affinity chromatography using Cry1Ab toxin coupled specificity of B. thuringiensis toxins correlates directly to Sepharose 4B. The apparent molecular mass of puriwith the presence of high-affinity binding proteins presfied soluble recombinant BT-R 1 is 195 kDa. Radiolaent in the midgut of target insects (7, 26), supporting the beled toxin bound to purified soluble BT-R 1 with a K d hypothesis (25) that insect receptor molecules mediate value of 1.1 nM, which is similar to that of both memtoxicity of these toxins. brane-bound BT-R 1 in Sf21 cells and natural BT-R 1 from A high-affinity protein receptor, BT-R 1 , for the B. M. sexta larval midgut tissue. Binding of radiolabeled thuringiensis Cry1A family of lepidopteran-specific toxtoxin to soluble BT-R 1 was competitively inhibited by ins has been identified (17, 24) and cloned (25) from unlabeled Cry1Ab toxin but not by other Cry toxins Manduca sexta larval midgut epithelial cells (4, 9, 10). as was observed also for membrane-bound BT-R 1. The BT-R 1 is a cadherin-related protein with an apparent recombinant soluble protein was stable in culture molecular weight of 210 kDa (24). Expression of BT-R 1 medium for at least 3 days at 27؇C and for 7 days at 4؇C is tissue specific. Only epithelial cells of M. sexta midgut and exhibited toxin-binding properties similar to the tissue harbor BT-R 1 mRNA and its translated protein natural protein. Apparently, neither membrane associ-(Midboe and Bulla, unpublished results). The cDNA for ation nor the extent of glycosylation influences the BT-R 1 (GenBank Accession No. AF319973) encodes a binding affinity and specificity of BT-R 1. Approxiprotein (25) comprising four putative domains: ectodomately 1 mg of purified BT-R 1 was obtained per liter main (EC 3 ; amino acid residues 1-1464), membraneof insect cell culture supernatant, representing ϳ2 ؋ proximal extracellular domain (MPED; residues 1465-10 9 Sf21 cells. ᭧ 2001 Academic Press 1532), a single transmembrane domain (residues 1533-1557), and cytoplasmic domain (residues 1558-1717).
Proceedings of the National Academy of Sciences, 2006
Many pathogenic organisms and their toxins target host cell receptors, the consequence of which i... more Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg 2+ -dependent cytotoxic event upon binding of the toxin to its receptor BT-R 1 . Here we show that binding of Cry toxin to BT-R 1 provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G αs ) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifest...
The objective of this study was to explore the role of molecular oxidative damage and caloric int... more The objective of this study was to explore the role of molecular oxidative damage and caloric intake in the aging process. The concentration of 8-hydroxydeoxyguanosine (8-OHdG), a product of DNA oxidation, was compared in five different tissues of mice (skeletal muscle, brain, heart, liver and kidney) as a function of age and in response to dietary restriction. A comparison of 8-and 27-month-old mice indicated that the age-related increase in 8-OHdG concentration was greater in skeletal muscle, brain and heart, which are primarily composed of long-lived, post-mitotic cells, than in liver and kidney, which consist of slowdividing cells. Dietary restricted (DR) mice kept on 60% caloric intake as compared to the ad libitum-fed (AL) mice showed a lower concentration in 8-OHdG content in all the tissues compared to AL mice. The DR-related amelioration of DNA oxidative damage was greater in the post-mitotic tissues compared to those undergoing slow mitoses. Results support the hypothesis that oxidative damage to long-lived post-mitotic cells may be a key factor in the aging process.
Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increa... more Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound ෂ60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of 125 I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K d ෂ0.1 µM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.
Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal p... more Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R 1 (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K d ෂ 1nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R 1 revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R 1 cDNA, subsequently killing the cells. Recruitment of BT-R 1 by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2003
The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R (210-kDa), which cont... more The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R (210-kDa), which contains 12 1 ectodomain modules in association with a number of motifs potentially involved in interactions with cadherin and integrin. The molecule is a target receptor for Bacillus thuringiensis Cry1A toxins that bind to BT-R with high affinity 1 and specificity. BT-R is localized exclusively in the midgut epithelium. The amount of BT-R protein increases 1 1 dramatically during larval development, paralleling accumulation of its mRNA. The 59-UTR of the btr-1 gene contains sequence motifs that most likely recruit specific transcription factors, particularly, those that determine posterior patterning and that control intestinal cell proliferation, differentiation and identity during development. The increase in abundance of BT-R may be required to support not only the differentiation of the epithelial cells but also the establishment of 1 physiological function and structural integrity of the midgut during larval development in M. sexta. We believe that BT-R is essential to larval midgut epithelial organization during rapid cell proliferation and tissue growth in M. sexta 1 because disruption of such organization and functionality occasioned by the binding of the Cry1A toxins of B. thuringiensis to BT-R causes death to the insect.
The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringi... more The specific role of cadherin receptors in cytotoxicity involving Cry toxins of Bacillus thuringiensis and their interactions with cell membrane has not been defined. To elucidate the involvement of toxin-membrane and toxinreceptor interactions in cytotoxicity, we established a cellbased system utilizing High Five insect cells stably expressing BT-R 1 , the cadherin receptor for Cry1Ab toxin. Cry1Ab toxin is incorporated into cell membrane in both oligomeric and monomeric form. Monomeric toxin binds specifically to BT-R 1 whereas incorporation of oligomeric toxin is nonspecific and lipid dependent. Toxin oligomers in the cell membrane do not produce lytic pores and do not kill insect cells. Rather, cell death correlates with binding of the Cry1Ab toxin monomer to BT-R 1 , which apparently activates a Mg 2 þdependent cellular signaling pathway.
The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding ... more The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R 1 , a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A
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