Biomedical subjects

D H Dean

Publications and source records attributed to D H Dean.

At least 37 records · Page 2Linked to original sources

PubMed1999

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.

Animals↗

PubMed1999

Evaluating the vocational rehabilitation program using longitudinal data. Evidence for a quasiexperimental research design.

The study presents benefit-cost ratios for 14 disability cohorts served by the Vocational Rehabilitation (VR) Program. The earnings impacts are estimated in a quasiexperimental framework using an internal comparison group. The earnings data are from a unique national panel constructed by linking client data of the Rehabilitative Services Administration with earnings histories from the Social Security Administration. These earnings data accommodate a series of statistical tests that allow us to identify and control for the presence of selection bias when estimating treatment impacts. The results indicate that the VR program is cost-effective in general, although not universally so across specific disabilities.

Adult↗

PubMed1998

Bacillus thuringiensis insecticidal proteins: molecular mode of action.

Growing interest in biorational pesticides has placed the Bacillus thuringiensis insecticidal crystal proteins at the forefront of pesticides for plant genetic engineering. The development of improvement pesticides, both in enhanced activity and broader host range, depends on an understanding of its mechanism of action. This review presents a complete overview of the bacterium and the group of insecticidal proteins known as Cry proteins or delta-endotoxins. The molecular mode of action is described in detail, including the mapping of receptor binding sites by site-directed mutagenesis, the known receptors, and the ion-channel activity of the toxins.

Amino Acid Sequence↗

PubMed1998

Functional complementation of nontoxic mutant binary toxins of Bacillus sphaericus 1593M generated by site-directed mutagenesis.

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.

Animals↗

PubMed1998

Bacillus thuringiensis and its pesticidal crystal proteins.

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Amino Acid Sequence↗

PubMed1998

Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.

The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.

Bacillus thuringiensis↗

PubMed1997

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.

Amino Acid Sequence↗

PubMed1997

Determination of Binding of Bacillus thuringiensis (delta)-Endotoxin Receptors to Rice Stem Borer Midguts.

Insecticidal activity and receptor binding properties of Bacillus thuringiensis toxins to yellow and striped rice stem borers (Sciropophaga incertulas and Chilo suppresalis, respectively) were investigated. Yellow stem borer (YSB) was susceptible to Cry1Aa, Cry1Ac, Cry2A, and Cry1C toxins with similar toxicities. To striped stem borer (SSB), Cry1Ac, Cry2A, and Cry1C were more toxic than Cry1Aa toxin. Binding assays were performed with (sup125)I-labeled toxins (Cry1Aa, Cry1Ac, Cry2A, and Cry1C) and brush border membrane vesicles (BBMV) prepared from YSB and SSB midguts. Both Cry1Aa and Cry1Ac toxins showed saturable, high-affinity binding to YSB BBMV. Cry2A and Cry1C toxins bound to YSB BBMV with relatively low binding affinity but with high binding site concentration. To SSB, both Cry1Aa and Cry1Ac exhibited high binding affinity, although these toxins are less toxic than Cry1C and Cry2A. Cry1C and Cry2A toxins bound to SSB BBMV with relatively low binding affinity but with high binding site concentration. Heterologous competition binding assays were performed to investigate the binding site cross-reactivity. The results showed that Cry1Aa and Cry1Ac recognize the same binding site, which is different from the Cry2A or Cry1C binding site in YSB and SSB. These data suggest that development of multitoxin systems in transgenic rice with toxin combinations which recognize different binding sites may be useful in implementing deployment strategies that decrease the rate of pest adaptation to B. thuringiensis toxin-expressing rice varieties.

Journal Article↗

PubMed1996

Protein engineering of Bacillus thuringiensis delta-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae.

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) approximately 4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (> 21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering delta-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Animals↗

PubMed1996

Involvement of two amino acid residues in the loop region of Bacillus thuringiensis Cry1Ab toxin in toxicity and binding to Lymantria dispar.

Two amino acids, Gly and Ser, at positions 282 and 283 in the loop region of domain II of Cry1Ab2 toxin are substituted with Ala and Leu in the Cry1Ab9-033 toxin. Cry1Ab2 exhibited about a 10-fold increase in toxicity and a 9-fold increase in binding affinity to Lymantria dispar compared to Cry1Ab9-033. However, these toxins showed similar toxicity and binding affinity to Manduca sexta and Spodoptera exigua. Heterologous competition assays and brush border membrane vesicle (BBMV) ligand blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxins recognized the same 210-kDa L dispar BBMV protein. No measurable differences in dissociation binding assays were observed between these two toxins. Digestion of these toxins with L dispar gut enzymes and BBMV proteases indicated no differences in stability. Ala and Leu residues in Cry1Ab9-033 were substituted with Gly and Ser by site-directed mutagenesis to produce mutant Cry1Ab alpha 8. This toxin exhibited full recovery of toxicity and binding affinity for L dispar. These data suggested that the residues Gly and Ser in the loop region might be directly involved in receptor binding and toxicity in L dispar.

Animals↗

PubMed1996

Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis--a minireview.

The current model of the mechanism of action of several Bacillus thuringiensis insecticidal crystal proteins (Cry) is reviewed and tested by site-directed mutagenesis experiments. Amino acid (aa) residues were substituted in each of the three domains of Cry toxins and the effects on toxin stability, binding to receptors, irreversible insertion into the membrane, and ion channel activity were examined. Mutant proteins with aa altered on the putative membrane-proximal surface of domain I are affected in insertion into the membrane and toxicity, but not in binding to the receptor. Alterations in the putative receptor-binding loops of domain II show an effect on the initial (reversible) binding to the receptor when certain aa are altered, while affecting irreversible binding when other aa are altered. Mutant proteins with aa altered in a conserved track of aa of domain III have altered ion channel properties, as measured by the voltage clamping of insect midguts and the K+ permeability of brush border membrane vesicles. In summary, domain I is involved in insertion into the membrane and affects ion channel function, domain II is involved in receptor binding and insertion into the membrane, and domain III is involved ion channel function, receptor binding, and insertion into the membrane.

Bacillus thuringiensis↗

PubMed1996

Mutations at domain II, loop 3, of Bacillus thuringiensis CryIAa and CryIAb delta-endotoxins suggest loop 3 is involved in initial binding to lepidopteran midguts.

Alanine substitutions of loop 3 residues, 438SGFSNS443, of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens. Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule. Bioassay data showed that mutant G439A (alanine substitution of residue Gly439) and F440A significantly reduced toxicity toward M. sexta and H. virescens. In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin. Binding studies with brush border membrane vesicles prepared from M. sexta and H. virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding. Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M. sexta and H. virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected. The role of hydrophobic residue, Phe440, is distinctly different from our previous observation that alanine substitution of Phe371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M. sexta (Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., and Dean, D. H. (1995) J. Bacteriol. 177, 2276-2282). Likewise, deletion of relatively hydrophobic CryIAa loop 3 residues, 440AAGA443 (D3a), resulted in reduced toxicity to Bombyx mori (>62 times less) and M. sexta (28 times less). The loss of toxicity was correlated with reduced initial binding to midgut vesicles prepared from these insects. However, alanine substitution of residues 437LSQ439 (A3a), contiguous to loop 3, altered neither toxicity nor receptor binding toward B. mori or M. sexta. These results suggest that the loop 3 residues of CryIAb and CryIAa toxins establish hydrophobic interactions with the receptor molecule, and mutations at these hydrophobic residues affect initial binding.

Amino Acid Sequence↗

PubMed1996

Substitution of residues on the proximal side of Cry1A Bacillus thuringiensis delta-endotoxins affects irreversible binding to Manduca sexta midgut membrane.

Substitution of a positively charged residue (R93F) or addition of a negatively charged residue (A92D) at the N-terminal of alpha 3 helix of domain I of the Cry1Ac delta-endotoxin resulted in a substantial reduction in toxicity against Manduca sexta. The N-terminal residues of helix 3 are considered to be on the same (proximal) surface of the toxin as the loops in domain II which are involved in the binding of the toxin to the receptor. The loss of toxicity was not caused by a decrease in the initial binding but rather by reduced irreversible binding. Only 65 and 75% of the A92D and R93F mutant toxin, respectively, bound to midgut vesicles irreversibly, compared to 94% of the wild type toxin. On the other hand, replacing A119 in a loop on the distal side of the helices with negatively charged residues (A119D or A119E) did not affect toxicity or irreversible binding.

Amino Acid Sequence↗

PubMed1996

Inconsistencies in determining Bacillus thuringiensis toxin binding sites relationship by comparing competition assays with ligand blotting.

Receptor binding properties of Cry1Aa, Cry1Ab, and Cry1Ac Bacillus thuringiensis toxins to Lymantria dispar brush border membrane vesicles (BBMV) were investigated by competition assays and BBMV ligand blotting. Homologous competition binding assays demonstrated that all Cry IA toxins bound to L. dispar BBMV with high binding affinities. Heterologous competition assays suggested that all three toxins share the same binding sites. However, our ligand blotting experiments were not consistent with the heterologous competition assays. We identified a 120 kDa peptide as a Cry1Ac binding protein and a 210 kDa peptide as a Cry1Aa and Cry1Ab binding protein. These results indicated that determining the relationships between toxin binding sites by either competition assays or ligand blotting alone may not be conclusive.

Animals↗

PubMed1996

Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA delta-endotoxin.

Analysis of the three surface loops in domain II of Bacillus thuringiensis CryIIIA delta-endotoxin has been carried out to assess their role in receptor binding and toxicity. Site-directed mutagenesis was used to convert loop residues to alanine and the mutant proteins were analyzed for structural stability, toxicity to beetle larvae (Tenebrio molitor), binding to receptors on T. molitor brush border membrane vesicles (Tm-BBMV) and insertion into BBMV, as measured by irreversible membrane receptor binding. This study demonstrates the functional significance of loops for binding and insertion. Alanine replacements in loop I resulted in disruption of receptor binding or structural instability. The double mutation, Y350A, Y351A, could be suppressed by replacing a nearby R345 with alanine, and the resultant mutant protein also showed reduced receptor binding. Substitution of N353 and D354 in loop I with alanine residues caused the loss of binding ability and toxicity. A loop II double mutant, P412A, S413A, had no effect on binding or toxicity. A block mutation of loop III residues to alanine had the effect of reducing receptor binding while concomitantly increasing toxicity by 2.4-fold. We compared this up-mutant to wild-type toxin in each step of physiological processing of protoxin: solubility, proteolytic activation, and insertion into the Tm-BBMV. The loop III block mutant showed increased membrane insertion, but was similar to wild-type toxin in other parameters. These results reveal that loop I and loop III in domain II of CryIIIA delta-endotoxin are involved in receptor binding. In addition, the direct correlation between toxicity and irreversible binding of the loop III block mutant (despite the indirect relationship to reversible binding) suggests that loop III may play a role in membrane insertion.

Animals↗

PubMed1996

Role of domain II, loop 2 residues of Bacillus thuringiensis CryIAb delta-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothis virescens.

Site-directed mutagenesis was used to examine the role of domain II, loop 2 residues, 368RRPFNIGI375, of Bacillus thuringiensis insecticidal protein CryIAb. Alanine substitution of residues 368RRP370, called B4, abolished potency toward Manduca sexta and Heliothis virescens, and the loss of toxicity was correlated directly to substantially reduced binding affinity to brush-border membrane vesicles (BBMV) prepared from the target insect midguts. These results indicated that these positive charges might be essential to orient the toxin to midgut receptor molecule(s). The role of residue Phe371 of CryIAb toxin to M. sexta was investigated by substituting a series of residues at this position. Irreversible binding and toxicity were affected significantly by hydrophilic, aliphatic, and smaller side-chain residues such as Cys, Val, Leu, and Ser but not by Tyr or Trp. A hydrophobic aromatic side-chain residue at position 371 was therefore essential for irreversible binding of CryIAb toxin in M. sexta. The role of residues 370PFNIGI375 of CryIAb toxin on H. virescens was also examined. Mutants D2 (deletion of residues 370-375), G374A (alanine substitution of Gly374), and I375A had reduced toxicity to H. virescens. In contrast to our findings with M. sexta, the reduction in toxicity of these mutants was correlated directly with loss of initial binding to H. virescens BBMV, indicating that these residues perform functionally distinct roles in binding and toxicity to different insects. In ligand blots, CryIAb recognized a major 210-kDa peptide in M. sexta BBMV and a 170-kDa peptide in H. virescens BBMV.

Amino Acid Sequence↗

PubMed1996

Site-directed mutations in the third domain of Bacillus thuringiensis delta-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles.

A series of mutant Bacillus thuringiensis CryIAa delta-endotoxin proteins was prepared by replacing the first, second, and last arginine residues of the conserved third-domain sequence, R-521 YRVRIR-527, with other amino acids. The stable mutant proteins were bioassayed against Bombyx mori larvae and found to all be approximately half as active as wild-type CryIAa. The toxins were also tested by means of a light-scattering assay for their ability to increase permeability of larval B. mori midgut brush border membrane vesicles. Three of the mutant toxins were as active as the wild-type toxin in the vesicle permeability assay.

Animals↗

PubMed1996

Synergistic effect of the Bacillus thuringiensis toxins CryIAa and CryIAc on the gypsy moth, Lymantria dispar.

The insecticidal activity of toxins CryIAa, CryIAb, and CryIAc against Lymantria dispar (gypsy moth) and Bombyx mori (silkworm) was examined by force-feeding bioassays. Toxin CryIAa exhibited higher toxicity than toxins CryIAb and CryIAc for L. dispar and B. mori. To evaluate possible synergism among these toxins, bioassays were performed with mixtures of CryIAa and CryIAb, CryIAb and CryIAc, and CryIAa and CryIAc. Expected toxicity was calculated from the activity of each individual toxin and its proportion in the mixture by using the equation described by Tabashnik (B. E. Tabashnik, Appl. Environ. Microbiol. 58:3343-3346, 1992). Observed 50% growth-inhibitory doses were calculated from mixing experiments by probit analysis. In L. dispar bioassays, synergism was observed with a mixture of CryIAa and CryIAc while a mixture of CryIAa and CryIAb exhibited an antagonistic effect. No synergistic effect on B. mori was observed with any toxin combination. Voltage clamping assays of isolated L. dispar midguts also demonstrated that the mixture of CryIAa and CryIAc induced a greater slope of inhibition of short circuit current than did other toxin combinations.

Animals↗