Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors.

BACKGROUND: To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. RESULTS: The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. CONCLUSIONS: These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin.

Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphate-linked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3'-pyrophosphate (P'-->5') adenosine (CppA) and uridine 3'-pyrophosphate (P'-->5') adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5'-AMP and cytidine or uridine cyclic 2',3'-phosphate. The k(cat)/K(m) values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K(m) values (10-16 microM) are lower than those reported for any earlier small substrates (e.g., 500-700 microM for CpA and UpA). The k(cat)/K(m) value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K(m) is more modest. Ribonucleotide 3',5'-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2',3'-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t(1/2) > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 degrees C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by approximately 10(5)-and 10(3.3)-fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5'-O leaving group, is not required for UppA cleavage because the pK(a) of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2'-deoxyuridine 3'-pyrophosphate (P'-->5') adenosine (dUppA), determined at 1.7 A resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through pi-pi stacking with the six-membered ring of adenine.

Mutations at the arginine residues in alpha8 loop of Bacillus thuringiensis delta-endotoxin Cry1Ac affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

The functional role of the alpha8 loop residues in domain II of Bacillus thuringiensis Cry1Ac toxin was examined. Alanine substitution mutations were introduced in the residues from 275 to 293. Among the mutant toxins, substitutions at R281 and R289 affected toxicity to Manduca sexta and Lymantria dispar. Loss of toxicity by these mutant toxins was well correlated with reductions in binding affinity for brush border membrane vesicles and the purified receptor, aminopeptidase N (APN), from both insects. These data suggest that the two arginine residues in the alpha8 loop region are important in toxicity and APN binding in L. dispar and M. sexta.

Low seasonal temperatures promote life cycle synchronization.

In this paper we discuss how seasonal temperature variation and life-stage specific developmental thresholds that cause quiescence can synchronize the seasonal development of exothermic organisms. Using a simple aging model it is shown that minimal seasonal temperature variation and periods of quiescence during extreme temperature conditions are sufficient to establish stable, univoltine ovipositional cycles. Quiescence induced by life-stage specific developmental thresholds, in fact, promotes synchronous oviposition and emergence. The mountain pine beetle, an important insect living in extreme temperature conditions and showing no evidence of diapause, invites direct application of this model. Simulations using mountain pine beetle parameters are used to determine temperature regimes for which stable ovipositional cycles exist.

Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity.

BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were futile, but it was degraded by proteases with broad specificity indicating the presence of a peptide. Carbohydrate was detected by labeling with digoxigenin hydrazide following periodate oxidation. Mild alkaline hydrolysis destroyed toxin and antibody binding, suggesting O-linked glycans are involved in the activity. GC/MS composition analysis showed that the predominant sugars were galactose, glucose, and N-acetyl galactosamine with lesser amounts of N-acetyl glucosamine, glucuronic acid, xylose, and fucose. The carbohydrate moiety accounted for 73% of its total mass. Amino acid analysis showed a high content of aspartic/asparagine, threonine, and serine residues in the protein moiety. The purified glycoconjugate was not visualized using Coomassie or silver staining procedures, but stained "blue" using the cationic dye Stains-all. BTR-270 was labeled with biotin and used as a diagnostic probe for screening and identifying toxins that bind to the receptor. Toxin-binding kinetics obtained using a biosensor demonstrated that the receptor binds Cry1Aa and Cry1Ab toxins with high affinity, and displays a weaker affinity for Cry1Ac, in correlation with the toxicity of these toxins towards gypsy moth. Arch.

Role of two arginine residues in domain II, loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis delta-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.

Two arginine residues (368-369) of Cry1Ab and Cry1Ac were mutated to alanine, glutamic acid and lysine by site-directed mutagenesis. Insecticidal activities of the mutant toxins on Manduca sexta and Lymantria dispar larvae were examined. Cry1Ac mutant toxins (c)RR-AA and (c)RR-EE and Cry1Ab mutant toxins (b)RR-AA and (b)RR-EE showed great reductions in toxicity against both insects. In contrast, conservatively changed (c)RR-KK and (b)RR-KK mutants did not alter toxicity to either insect. Binding assays with brush border membrane vesicles (BBMVs) prepared from L. dispar midguts demonstrated that (c)RR-AA, (c)RR-EE, (b)RR-AA and (b)RR-EE bound with lower affinities compared with their respective wild-type toxins. To M. sexta BBMVs, (c)RR-AA and (c)RR-EE showed great reductions in BBMV binding. However, (b)RR-AA and (b)RR-EE did not alter BBMV competition patterns, despite their reduced toxicity. Further binding assays were performed with aminopeptidase N (APN) purified from L. dispar and M. sexta BBMVs using surface plasmon resonance (BIAcore). Direct correlation between toxicity and APN binding was observed for the mutant toxins using this technique. The inconsistency between BBMV and APN binding data with Cry1Ab to M. sexta suggests the possibility of a different Cry1Ab toxin-binding mechanism or the importance of another receptor in M. sexta.

Seasonal temperature alone can synchronize life cycles.

In this paper we discuss the effects of yearly temperature variation on the development and seasonal occurrence of poikiliothermic organisms with multiple life stages. The study of voltinism in the mountain pine beetle (Dendroctonus ponderosae Hopkins), an important forest insect living in extreme temperature environments and exhibiting no diapause, provides a motivational example. Using a minimal model for the rates of aging it is shown that seasonal temperature variation and minimal stage-specific differences in rates of aging are sufficient to create stable uni- and multi-voltine oviposition cycles. In fact, these cycles are attracting and therefore provide an exogenous mechanism for synchronizing whole populations of organisms. Structural stability arguments are used to extend the results to more general life systems.

Bivalent sequential binding model of a Bacillus thuringiensis toxin to gypsy moth aminopeptidase N receptor.

Specificity for target insects of Bacillus thuringiensis insecticidal Cry toxins is largely determined by toxin affinity for insect midgut receptors. The mode of binding for one such toxin-receptor complex was investigated by extensive toxin mutagenesis, followed by real-time receptor binding analysis using an optical biosensor (BIAcore). Wild-type Cry1Ac, a three-domain, lepidopteran-specific toxin, bound purified gypsy moth (Lymantria dispar) aminopeptidase N (APN) biphasically. Site 1 displayed fast association and dissociation kinetics, while site 2 possessed slower kinetics, yet tighter affinity. We empirically determined that two Cry1Ac surface regions are involved in in vivo toxicity and APN binding. Mutations within domain III affected binding rates to APN site 1, whereas mutations in domain II affected binding rates to APN site 2. Furthermore, domain III contact is completely inhibited in the presence of N-acetylgalactosamine, indicating loss of domain III binding eliminates all APN binding. Based upon these observations, the following model is proposed. A cavity in lectin-like domain III initiates docking through recognition of an N-acetylgalactosamine moiety on L. dispar APN. Following primary docking, a higher affinity domain II binding mechanism occurs, which is critical for insecticidal activity.

Effects of 2,4,6-trinitrotoluene in a holistic environmental exposure regime on a terrestrial salamander, Ambystoma tigrinum.

2,4,6-Trinitrotoluene (TNT) is a defense-related environmental contaminant present at high concentrations in soil at some military installations. Tiger salamanders (Ambystoma tigrinum, family Ambystomatidae) were exposed to TNT in a soil matrix and fed earthworms that had also been exposed to TNT via contaminated soil. Such exposure was previously shown to result in significant accumulation of both TNT and TNT metabolites by salamanders. Following 14 days of combined oral and dermal exposures, salamanders were evaluated for signs of toxicity. Control and TNT-exposed salamanders gained weight (p < 0.025). In addition, organ to body weight ratios (kidney, liver, and spleen) were not affected by treatment. The function of splenic phagocytic cells was evaluated because these cells are sensitive to certain environmental chemical exposures. Neither the chemiluminescence response (H2O2 production) nor the phagocytic capacity of such cells were different between controls and treatment groups. In like manner, no changes were seen in the peripheral hematologic parameters investigated. Histopathologic evaluations were inconclusive, yet the liver revealed the presence of heavily pigmented iron-rich phagocytes (melanomacrophages). This investigation presents a realistic approach and preliminary data for investigating the effects of xenobiotic exposure in a soil matrix on a terrestrial vertebrate.

Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase-N receptor is not directly related to toxicity.

Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds a 115-kDa aminopeptidase-N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar-binding pocket and binding to purified aminopeptidase-N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509-511 (QNR-AAA) eliminated aminopeptidase-N binding and reduced binding to BBMV. However, toxicity decreased no more than two-fold, indicating activity is not directly correlated with aminopeptidase-N binding. Analysis of toxin binding to aminopeptidase-N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.

The corporate medical department and AIDS.

HIV infection in the workplace reflects the dimensions and distribution of the problem in the community at large. Despite this, accommodation of ill employees has been the general pattern and disruptions of any kind the exception. Corporate medical departments can be important participants in the clinical and social response to AIDS. Recognition of the illness itself, collaboration in the provision of health services, referral when necessary for treatment and counselling, assistance with medical benefits, and arranging for appropriate workplace modifications are among ways in which medical departments can assist concerned or ill employees. Confidentiality is essential in all of these interactions. Education of the workforce about AIDS is especially important from a public health as well as from an administrative standpoint. Providing input on relevant corporate policies and in determination of corporate philanthropy can be other dimensions of corporate medical departments' response to AIDS.