Comparison of different methodologies for binding assays of Bacillus thuringiensis toxins to membrane vesicles from insect midguts.
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Animals↗
Biomedical subjects
S Herrero
Publications and source records attributed to S Herrero.
Variation in susceptibility to Bacillus thuringiensis toxins among unselected strains of Plutella xylostella.
So far, the only insect that has evolved resistance in the field to Bacillus thuringiensis toxins is the diamondback moth (Plutella xylostella). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to B. thuringiensis toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to B. thuringiensis toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five B. thuringiensis toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.
Animals↗
High genetic variability for resistance to Bacillus thuringiensis toxins in a single population of diamondback moth.
The long-term benefit of insecticidal products based on Cry toxins, either in sprays or as transgenic crops, is threatened by the development of resistance by target pests. The models used to predict evolution of resistance to Cry toxins most often are monogenic models in which two alleles are used. Moreover, the high-dose/refuge strategy recommended for implementation with transgenic crops relies on the assumption that the resistance allele is recessive. Using selection experiments, we demonstrated the occurrence in a laboratory colony of diamondback moth of two different genes (either allelic or nonallelic) that confer resistance to Cry1Ab. At the concentration tested, resistance was dominant in one selection line and partially recessive in the other. Resistant insects from the two selection lines also differed in their cross-resistance patterns. The diamondback moth colony was derived from a field population from the Philippines, which originally showed a different resistance phenotype. This is the first time that an insect population has been directly shown to carry more than one gene conferring resistance to the same Cry toxin.
Animals↗
Shared binding sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A toxins.
Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At least two strains of diamondback moth (Plutella xylostella) with resistance to Cry1A toxins and reduced binding of Cry1A toxins have strong cross-resistance to Cry1Ja. Thus, we hypothesized that Cry1Ja shares binding sites with Cry1A toxins. We tested this hypothesis in six moth and butterfly species, each from a different family: Cacyreus marshalli (Lycaenidae), Lobesia botrana (Tortricidae), Manduca sexta (Sphingidae), Pectinophora gossypiella (Gelechiidae), P. xylostella (Plutellidae), and Spodoptera exigua (Noctuidae). Although the extent of competition varied among species, experiments with biotinylated Cry1Ja and radiolabeled Cry1Ac showed that Cry1Ja and Cry1Ac competed for binding sites in all six species. A recent report also indicates shared binding sites for Cry1Ja and Cry1A toxins in Heliothis virescens (Noctuidae). Thus, shared binding sites for Cry1Ja and Cry1A occur in all lepidopteran species tested so far.
Amino Acid Sequence↗
Mannose phosphate isomerase isoenzymes in Plutella xylostella support common genetic bases of resistance to Bacillus thuringiensis toxins in Llpidopteran species.
A strong correlation between two mannose phosphate isomerase (MPI) isoenzymes and resistance to Cry1A toxins from Bacillus thuringiensis has been found in a Plutella xylostella population. MPI linkage to Cry1A resistance had previously been reported for a Heliothis virescens population. The fact that the two populations share similar biochemical, genetic, and cross-resistance profiles of resistance suggests the occurrence of homologous resistance loci in both species.
Animals↗
Different mechanisms of resistance to Bacillus thuringiensis toxins in the indianmeal moth.
Susceptibility to protoxin and toxin forms of Cry1Ab and the binding of (125)I-labeled Cry1Ab and Cry1Ac has been examined in three Plodia interpunctella colonies, one susceptible (688(s)) and two resistant (198(r) and Dpl(r)) to Bacillus thuringiensis. Toxicological studies showed that the 198(r) colony was 11-fold more resistant to Cry1Ab protoxin than to Cry1Ab activated toxin, whereas the Dpl(r) colony was 4-fold more resistant to protoxin versus toxin. Binding results with (125)I-labeled toxins indicated the occurrence of two different binding sites for Cry1Ab in the susceptible insects, one of them shared with Cry1Ac. Cry1Ab binding was found to be altered in insects from both resistant colonies, though in different ways. Compared with the susceptible colony, insects from the Dpl(r) colony showed a drastic reduction in binding affinity (60-fold higher K(d)), although they had similar concentrations of binding sites. Insects from the 198(r) colony showed a slight reduction in both binding affinity and binding site concentration (five-fold-higher K(d) and ca. three-fold-lower R(t) compared with the 688(s) colony). No major difference in Cry1Ac binding was found among the three colonies. The fact that the 198(r) colony also has a protease-mediated mechanism of resistance (B. Oppert, R. Hammel, J. E. Throne, and K. J. Kramer, J. Biol. Chem. 272:23473-23476, 1997) is in agreement with our toxicological data in which this colony has a different susceptibility to the protoxin and toxin forms of Cry1Ab. It is noteworthy that the three colonies used in this work derived originally from ca. 100 insects, which reflects the high variability and high frequency of B. thuringiensis resistance genes occurring in natural populations.
Animals↗
Alkylpyridine complexes of tungsten(II) and chromium(II). First rotational isomers of W2X4L4 molecules with D2h and D2 symmetries.
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Journal Article↗
Mixed chloride/amine complexes of dimolybdenum(II,II). 6. Stepwise substitution of amines by tertiary phosphines and vice versa: stereochemical hysteresis.
The substitution reactions of primary amines by tertiary phosphines in quadruply bonded dimolybdenum(II,II) complexes Mo2Cl4(NH2R)4 have been studied. The exchange reaction has been shown to result at room temperature in disubstituted species Mo2Cl4(NH2R)2(PR3)2 (PR3 = PMe3, NH2R = NH2Prn (1a), NH2But (2a), NH2Cy (3a); PR3 = PMe2Ph, NH2R = NH2Cy (4a)), while heating is needed to obtain fully substituted complexes Mo2Cl4(PR3)4. The crystal structure of disubstituted products has been investigated by X-ray crystallography and revealed that they all belong to the alpha-isomer, having both phosphine groups at the same Mo atom. Crystal data are as follows: for 1a, tetragonal space group I4(1/a) with a = 17.737(2) A, c = 15.6915(6) A, and Z = 8; for 3a, monoclinic space group P2(1) with a = 10.963(3) A, b = 10.117(2) A, c = 13.323(4) A, beta = 90.05(2) degrees, and Z = 2; for 4a, triclinic space group P1 with a = 9.329(3) A, b = 10.206(2) A, c = 18.975(3) A, alpha = 85.45(2) degrees, beta = 87.10(1) degrees, gamma = 80.88(1) degrees, and Z = 2. The substitution processes for the direct and reverse reactions have been monitored by 31P NMR. They both proceed in a stepwise manner; however, a stereochemical hysteresis is taking place, i.e., the back reaction, the substitution of phosphines by amines, goes through another isomer of Mo2Cl4(NH2R)2(PR3)2, having phosphine ligands on different Mo atoms. This beta-isomer is more thermodynamically stable and can be obtained by thermal conversion of the alpha-form. All chemical equilibria studied in the paper have been explained as governed by a higher trans effect of PR3 groups compared to NH2R groups.
Journal Article↗
Genetic and biochemical approach for characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in a field population of the diamondback moth, Plutella xylostella.
Four subpopulations of a Plutella xylostella (L.) strain from Malaysia (F(4) to F(8)) were selected with Bacillus thuringiensis subsp. kurstaki HD-1, Bacillus thuringiensis subsp. aizawai, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F(9) found that selection with Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai in UNSEL-MEL declined significantly by F(9). The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab, B. thuringiensis subsp. kurstaki, and B. thuringiensis subsp. aizawai (5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-, B. thuringiensis subsp. kurstaki-, and B. thuringiensis subsp. aizawai-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F(9) to F(13)) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with (125)I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F(15)). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F(2) progeny from a backcross of F(1) progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.
Animals↗
Development and characterization of diamondback moth resistance to transgenic broccoli expressing high levels of Cry1C.
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 selection but decreased to 235-fold at G38 when selection ceased at G28. The Cry1C resistance in this strain was seen to be inherited as an autosomal and incompletely recessive factor or factors when evaluated using a leaf dip assay and recessive when evaluated using Cry1C transgenic broccoli. Saturable binding of (125)I-Cry1C was found with brush border membrane vesicles (BBMV) from both susceptible and Cry1C-resistant strains. Significant differences in Cry1C binding to BBMV from the two strains were detected. BBMV from the resistant strain had about sevenfold-lower affinity for Cry1C and threefold-higher binding site concentration than BBMV from the susceptible strain. The overall Cry1C binding affinity was just 2.5-fold higher for BBMV from the susceptible strain than it was for BBMV from the resistant strain. These results suggest that reduced binding is not the major mechanism of resistance to Cry1C.
Animals↗
The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease.
We have analysed the quantitative expression of surface CD69 antigen on human mast cells (MC), from both normal and pathological bone marrow (BM) samples, using flow cytometry. Our major aim was to analyse whether CD69 is constitutively expressed by normal BMMC and to explore the possible differences between CD69 expression by BMMC from normal controls and patients suffering from different pathological conditions. The constitutive expression of surface CD69 was clearly demonstrated in BMMC; however, systemic mast cell disease (SMCD) patients showed significantly higher levels of surface CD69 expression than healthy controls (P < 0.001), chronic lymphocytic leukaemia (P = 0.001), monoclonal gammopathy of unknown significance (P < 0.001), multiple myeloma (P < 0.001) patients, and myelodysplastic syndromes (P = 0.002). Furthermore, almost no overlap between the levels of CD69 expression on BMMC was observed between SMCD cases and the remaining groups of individuals except for the paediatric mastocytosis group (P > 0.05). From the other groups of patients, monoclonal gammopathy of unknown significance (P = 0.04), myelodysplastic syndromes (P = 0.03) and paediatric mastocytosis (P = 0.003) cases showed a significantly higher expression of surface CD69 as compared to normal subjects. In summary, our findings show that the CD69 antigen is overexpressed in SMCD patients.
Adolescent↗
Characterization of the hemin-sensitive eukaryotic initiation factor 2alpha kinase from mouse nonerythroid cells.
The heme-regulated eukaryotic initiation factor 2alpha (eIF2alpha) kinase (heme-regulated inhibitor (HRI)) is activated by heme deficiency in reticulocytes and plays an important role in translational control in these cells. Previously, HRI was cloned from rabbit reticulocytes and rat brain, but a heme-regulated eIF2alpha kinase activity has only been purified from erythroid cells. In this study, we report the purification of a heme-sensitive eIF2alpha kinase activity from both mouse liver and NIH 3T3 cell extracts. Furthermore, we have cloned and characterized this mouse liver eIF2alpha kinase (mHRI), which exhibits 83 and 94% identities to rabbit and rat HRIs, respectively. Both the purified enzyme and recombinant mHRI exhibited an autokinase and an eIF2alpha kinase activity, and both activities were inhibited in vitro by hemin. In addition, wild-type mHRI, but not the inactive mHRI-K196R mutant, was autophosphorylated in vivo when it was expressed in 293 cells. Quantitation of mHRI mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction revealed relatively high levels in liver, kidney, and testis. These results provide strong evidence that mHRI is a ubiquitous eIF2alpha kinase of mammalian cells, suggesting that it could play important roles in the translational regulation of nonerythroid tissues.
Amino Acid Sequence↗
[Von Willebrand disease: characteristics and response to desmopressin. Study of 103 cases].
BACKGROUND: To describe the main characteristics and response to desmopressin infusion in 103 patients suffering from von Willebrand disease (vWD). PATIENTS AND METHODS: The criteria for diagnosis were (except for type 2N) the coexistence of von Willebrand factor ristocetin cofactor (vWF:RCo) activity < 50 U/dl with bleeding disease or one of the following data: von Willebrand factor antigen (vWF:Ag) activity < 50 U/dl, factor VIII (FVIII) activity < 50 U/dl or the existence of a increased bleeding time (BT). Multimeric studies of vWF were performed in 51 cases and ristocetin induced platelet aggregation (RIPA) was also performed. RESULTS: Spontaneous bleeding was found in 36 patients, while in 18 cases the diagnosis was done after surgical bleeding. Thirteen patients (6 presenting with mild bleeding) were studied for abnormalities in the routine preanestesic tests. Other 22 patients were diagnosed with vWD by familial studies. There were 3 patients with type 2B, 1 case with type 2N and other patient with type 3. BT was found increased in 26 out of 58 patients. The activities of vWF:CoR and vWF:Ag were 38.4 (9.4) U/dl and 45.8 (23.2) U/dl, respectively, while the activity of FVIII was 49.9 (20.8) U/dl. Prophylactic DDAVP (desmopressin) was infused in 32 patients. After 1 h, basal activities of vWF:CoR and vWF:Ag were increased by 3.1 (3.2) and 3.4 (3.1) times, respectively, and maintained for 3 h. FVIII activity increased 3.6 (2.3) times the basal levels decreasing after 3 h (2.9 [2.1]; p < 0.01). The BT was corrected in 8 out of ten patients. CONCLUSIONS: vWD is a major cause of surgical bleeding. Preanestesic anamnesis and coagulation tests can be useful to identify vWD. Many patients with vWD have normal BT. A failure in the response to desmopressin infusion is unusual.
Deamino Arginine Vasopressin↗
Human bone marrow mast cells from indolent systemic mast cell disease constitutively express increased amounts of the CD63 protein on their surface.
The quantitative measurement of the expression of both cytoplasmic and surface CD63 antigen by human mast cells from both normal and pathological bone marrow samples was studied by use of flow cytometry. Our major goal was to analyze whether in vivo CD63 expression by human bone marrow mast cells could be useful to discriminate bone marrow mast cells from patients with mastocytosis from other conditions. For that purpose, a total of 65 subjects corresponding to 12 healthy volunteers, 25 B-cell chronic lymphoproliferative disorders, 5 reactive bone marrow samples, 4 myelodysplastic syndromes, and 19 mastocytosis were analyzed. The expression of both surface and cytoplasmic CD63 by human bone marrow mast cells is clearly demonstrated. Our results show that high amounts of CD63 are present in human bone marrow mast cells most of it corresponding to an intracellular localization. No significant differences in CD63 expression were observed as regards both total and cytoplasmic CD63, except for higher CD63 levels in adult patients with mastocytosis (P = 0.05). By contrast, the mean level of surface CD63 significantly varied between the different groups of individuals. Accordingly, patients with monoclonal gammopathies displayed a slight decrease (P = 0.1) in surface CD63 expression, whereas bone marrow mast cells from adults with indolent systemic mast cell disease showed significantly (P = 0.0005) higher levels of surface CD63 as compared to healthy controls.
Adult↗
Low bone density with normal bone turnover in ovariectomized and streptozotocin-induced diabetic rats.
Diabetes and estrogen deficit are known causes of osteopenia, diabetes being associated with a low bone turnover and estrogen deficit with a high bone turnover. In the present work, we studied the effect of combined ovariectomy and diabetes on bone mineral content (BMC) and bone mineral density (BMD) and several bone markers in the rat. Four groups of rats were studied: control (C), ovariectomized (O), diabetic (D), and ovariectomized and diabetic (DO). Twelve weeks after starting the experiments, BMC and BMD of the first six lumbar vertebrae were measured; a bone formation marker (BGP) and a bone resorption marker (free collagen cross-links, PYD) were also analyzed. Diabetic rats showed diminished gain in bone mass, BMC (D: 0.417 +/- 0.028 g, DO: 0.422 +/- 0.020 g) and BMDs (D: 0.171 +/- 0.006 g/cm2, DO: 0.174 +/- 0.006 g/cm2) both being significantly (P < 0.001) lower than those of control (C: BMC 0.727 +/- 0.024 g and BMD 0.258 +/- 0.004 g/cm2) and ovariectomized (O: BMC 0.640 +/- 0.044 g and BMD 0.240 +/- 0.009 g/cm2) groups. Moreover, the BMC and BMD of the C group were significantly (P < 0.05) higher than that of the O group. BGP and PYD levels were significantly (P < 0.01) higher in the O group (BGP: 138.2 +/- 16.8 ng/ml, PYD: 270.2 +/- 17.8 nM/mM) than those found in the control rats (BGP: 44.7 +/- 4.8 ng/ml, PYD: 165.6 +/- 12.5 nM/mM); the D group showed significantly (P < 0.01) lower values (BGP: 27.4 +/- 14.6 ng/ml, PYD: 55.0 +/- 7.4 nM/mM) than those of the control group. The DO group showed similar levels (BGP: 43.4 +/- 5.1 ng/ml, PYD: 146.7 +/- 14.6 nM/mM) to those found in the C group. Although bone marker levels in the O and D groups were in accordance with those expected in these situations, in the DO group the corresponding levels are apparently "normal." Also, the decrease of gain in bone mass observed after combining estrogen deficit and diabetes (DO group) did not seem to be more marked than that caused by diabetes alone.
Amino Acids↗
Mantle cell lymphoma with amyotrophic lateral sclerosis (motor neuron disease)
We describe a previously unreported case of mantle cell lymphoma (MCL) associated to amyotrophic lateral sclerosis (ALS) in a 63-year-old woman with a 1-year history of weakness of arm and leg muscles. The both molecular-genetic and flow cytometry analysis of lymphocytes of peripheral blood (PB) demonstrated leukemic phase of MCL.
Amyotrophic Lateral Sclerosis↗
New trifluoromethyl ketones as potent inhibitors of esterases: 19F NMR spectroscopy of transition state analog complexes and structure-activity relationships.
A variety of trifluoromethyl ketones (TFMKs) have been studied as inhibitors of the antennal esterases of the Egyptian armyworm Spodoptera littoralis. The chemicals behaved as tight slow-binding inhibitors, the beta-thio derivatives being the most potent ones, particularly 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) with an IC50 of 0.08 microM. Other TFMKs, containing a propenyl group at the non-polar end of the molecule as in the natural pheromone structure (compounds 4, 8 and 9), were also notably active. Correlation studies of inhibition with lipophilicity (log P) indicated that the optimum log P values for activity of the beta-thio compounds are in the range of 3.03-5.11, while a higher lipophilicity (range 5.37-5.89) was required among the devoid of sulfur ketones. 19F NMR studies, carried out with OTFP, showed that the inhibitor binds the enzyme in a reversible manner, forming an adduct (probably a hemiacetal) of tetrahedral geometry with the active site of the enzyme. To our knowledge, this is the first time that such a study on a natural esterase extract has been undertaken.
Acetone↗
Heterogeneous decrease of bone mineral density in the vertebral column of ovariectomized rats.
The long-term effect of ovariectomy on the loss of bone mineral density (BMD) was evaluated in rats with and without estrogen treatment; BMD was studied in the lumbar and caudal vertebrae, measured by DXA, to find how the losses of BMD occur in the axial skeleton. Seventy female Wistar rats of 3 months of age were divided into four groups as follows: group 1: control animals; group 2: ovariectomized animals; group 3: ovariectomized animals undergoing treatment with estrogen (0.25 mg/kg per week of 17-beta estradiol); group 4: ovariectomized rats undergoing estrogen treatment only during the last 3 months of the experimental period. No significant differences were found among the groups in regard to the BMD values of the caudal vertebrae at either 3 or 6 months. Likewise, in the lumbar vertebrae there were no significant differences among the groups after 3 months. However, at 6 months, a decrease in the BMDs of the ovariectomized animals with respect to the remaining groups was found: 226 +/- 11 mg/cm2 in the ovariectomized group; 262 +/- 14 mg/cm2 in the controls; 255 +/- 4 mg/cm2 in the rats receiving estrogen treatment for 6 months; and 259 +/- 5 mg/cm2 in the animals receiving estrogen for 3 months. The study also reveals the absence of differences in the bone mineral density between the ovariectomized and control rats when the former received estrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Absorptiometry, Photon↗