Biomedical subjects

Isabel Gómez

Publications and source records attributed to Isabel Gómez.

18 recordsLinked to original sources

PubMed2006

Theoretical investigation of luminescence behavior as a function of alkyl chain size in 4-aminobenzonitrile alicyclic derivatives.

There is still controversy about the structure of the intramolecular charge transfer (ICT) emitting species in pi-electron donor-acceptor systems that show dual fluorescence. Although the twisted ICT model is quite generally accepted, the planar ICT model is not ruled out because firm experimental evidence supports it. Among these it is the fact that some rigidized systems such as bicyclic 4-aminobenzonitrile derivatives exhibit dual fluorescence. We present here an ab initio CASSCF/CASPT2 study of a series of these compounds with the alicyclic chain ranging from 5 to 7 carbon atoms and compare their ICT mechanism with the more flexible 4-aminobenzonitrile (ABN) and 4-(dimethylamino)benzonitrile (DMABN). We present the energetics, geometries, and valence bond structures of the critical points of the potential-energy surfaces of the ground, local excited (LE), and ICT states. Our results show that the photophysical differences of the studied systems may be rationalized by two factors: the position of the ICT and LE potential-energy surfaces at the first stages of the ICT reaction and the relative energies of the excited-state minima. Computational evidence is presented that a twisted ICT structure can be adopted in some molecules such as NXC6 and NXC7 and that the anomalous band of the fluorescence spectra of these systems is emitted from a twisted ICT species.

Journal Article↗

PubMed2006

Specific epitopes of domains II and III of Bacillus thuringiensis Cry1Ab toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta.

The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or beta16-beta22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III beta16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through beta-16 of domain III promoting membrane insertion and cell death.

Animals↗

PubMed2006

Structural changes of the Cry1Ac oligomeric pre-pore from bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion.

The primary action of Cry toxins produced by Bacillus thuringiensis is to lyse midgut epithelial cells in their target insect by forming lytic pores. The toxin-receptor interaction is a complex process, involving multiple interactions with different receptor and carbohydrate molecules. It has been proposed that Cry1A toxins sequentially interact with a cadherin receptor, leading to the formation of a pre-pore oligomer structure, and that the oligomeric structure binds to glycosylphosphatidyl-inositol-anchored aminopeptidase-N (APN) receptor. The Cry1Ac toxin specifically recognizes the N-acetylgalactosamine (GalNAc) carbohydrate present in the APN receptor from Manduca sexta larvae. In this work, we show that the Cry1Ac pre-pore oligomer has a higher binding affinity with APN than the monomeric toxin. The effects of GalNAc binding on the toxin structure were studied in the monomeric Cry1Ac, in the soluble pre-pore oligomeric structure, and in its membrane inserted state by recording the fluorescence status of the tryptophan (W) residues. Our results indicate that the W residues of Cry1Ac have a different exposure to the solvent when compared with that of the closely related Cry1Ab toxin. GalNAc binding specifically affects the exposure of W545 in the pre-pore oligomer in contrast to the monomer where GalNAc binding did not affect the fluorescence of the toxin. These results indicate a subtle conformational change in the GalNAc binding pocket in the pre-pore oligomer that could explain the increased binding affinity of the Cry1Ac pre-pore to APN. Although our analysis did not reveal major structural changes in the pore-forming domain I upon GalNAc binding, it showed that sugar interaction enhanced membrane insertion of soluble pre-pore oligomeric structure. Therefore, the data presented here permits to propose a model in which the interaction of Cry1Ac pre-pore oligomer with APN receptor facilitates membrane insertion and pore formation.

Acetylglucosamine↗

PubMed2006

Structural and functional analysis of the pre-pore and membrane-inserted pore of Cry1Ab toxin.

Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.

Bacillus thuringiensis↗

PubMed2006

Functional display of Bacillus thuringiensis Cry1Ac toxin on T7 phage.

The Cry1Ac toxin from Bacillus thuringiensis was displayed on the surface of T7 phage. The cry1Ac gene was fused to the C-terminal end of T7-10B capsid protein and displayed on the surface of T7 phage as revealed by Western blot analysis of the purified phage particles. The T7-Cry1Ac phages retained toxicity against Manduca sexta larvae. We demonstrated that the T7-Cry1Ac phage interacts with Cry1Ac receptors present in M. sexta BBMV either in solution or in overlay binding assays.

Animals↗

PubMed2006

Efficient photochemical merocyanine-to-spiropyran ring closure mechanism through an extended conical intersection seam. A model CASSCF/CASPT2 study.

A mechanism of the thermal and photochemical bleaching of merocyanine to spiropyran is proposed on the basis of CASSCF/CASPT2 calculations on the 6-(2-propenyliden)cyclohexadienone model system. Our results suggest that this photochemical transformation takes place in two steps. First, the initially pumped 1(pi-pi) S2 undergoes radiationless decay to 1(n-pi) S1 via an extended S2/S1 conical intersection seam that runs approximately parallel to the trans-to-cis isomerization coordinate, a few kilocalories per mole higher in energy. Thus, S2 --> S1 internal conversion is possible at all values of the S2 trans-to-cis reaction coordinate. Second, on the S1 potential energy surface, there is a barrierless ring closure reaction path from the S1 cis minimum that leads to a peaked S1/S0 conical intersection where the deactivation to the ground state takes place. The inertia of the moving nuclei then drives the system toward the ground-state minimum of the 2H-chromene product. Thus, the extended seam topology of the S2/S1 conical intersection and the coordinate of the branching space of the S1/S0 conical intersection are essential to explain the efficiency and high speed of this reaction.

Journal Article↗

PubMed2006

Role of tryptophan residues in toxicity of Cry1Ab toxin from Bacillus thuringiensis.

Bacillus thuringiensis produces insecticidal proteins (Cry protoxins) during the sporulation phase as parasporal crystals. During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins which form ionic pores. The structural changes of Cry toxins during oligomerization and insertion into the membrane are still unknown. The Cry1Ab toxin has nine tryptophan residues; seven are located in domain I, the pore-forming domain, and two are located in domain II, which is involved in receptor recognition. Eight Trp residues are highly conserved within the whole family of three-domain Cry proteins, suggesting an essential role for these residues in the structural folding and function of the toxin. In this work, we analyzed the role of Trp residues in the structure and function of Cry1Ab toxin. We replaced the Trp residues with phenylalanine or cysteine using site-directed mutagenesis. Our results show that W65 and W316 are important for insecticidal activity of the toxin since their replacement by Phe reduced the toxicity against Manduca sexta. The presence of hydrophobic residue is important at positions 117, 219, 226, and 455 since replacement by Cys affected either the crystal formation or the insecticidal activity of the toxin in contrast to replacement by Phe in these positions. Additionally, some mutants in positions 219, 316, and 455 were also affected in binding to brush border membrane vesicles (BBMV). This is the first report that studies the role of Trp residues in the activity of Cry toxins.

Animals↗

PubMed2005

A nuclear gene for the iron-sulfur subunit of mitochondrial complex II is specifically expressed during Arabidopsis seed development and germination.

Three nuclear genes, SDH2-1, SDH2-2 and SDH2-3, encode the essential iron-sulfur subunit of mitochondrial complex II in Arabidopsis thaliana. SDH2-1 and SDH2-2 probably arose via a recent duplication event and we reported that both are expressed in all organs from adult plants. In contrast, transcripts from SDH2-3 were not detected. Here we present data demonstrating that SDH2-3 is specifically expressed during seed development. SDH2-3 transcripts appear during seed maturation, persist through desiccation, are abundant in dry seeds and markedly decline during germination. Analysis of transgenic Arabidopsis plants carrying the SDH2-3 promoter fused to the beta-glucuronidase reporter gene shows that the SDH2-3 promoter is activated in the embryo during maturation, from the bent-cotyledon stage. beta-Glucuronidase expression correlates with the appearance of endogenous SDH2-3 transcripts, suggesting that control of this nuclear gene is achieved through transcriptional regulation. Furthermore, progressive deletions of this promoter identified a 159 bp region (-223 to -65) important for SDH2-3 transcriptional activation in seeds. Interestingly, the SDH2-3 promoter remains active in embryonic tissues during germination and post-germinative growth, and is turned off in vegetative tissues (true leaves). In contrast to SDH2-3 transcripts, SDH2-1 and SDH2-2 transcripts are barely detected in dry seeds and increase during germination and post-germinative growth. The opposite expression patterns of SDH2 nuclear genes strongly suggest that during germination the embryo-specific SDH2-3 is replaced by SDH2-1 or SDH2-2 in mitochondrial complex II.

Arabidopsis↗

PubMed2005

Intramolecular charge transfer in 4-aminobenzonitriles does not necessarily need the twist.

In electron donor/acceptor species such as 4-(dimethylamino)benzonitrile (DMABN), the excitation to the S(2) state is followed by internal conversion to the locally excited (LE) state. Dual fluorescence then becomes possible from both the LE and the twisted intramolecular charge-transfer (TICT) states. A detailed mechanism for the ICT of DMABN and 4-aminobenzonitrile (ABN) is presented in this work. The two emitting S(1) species are adiabatically linked along the amino torsion reaction coordinate. However, the S(2)/S(1) CT-LE radiationless decay occurs via an extended conical intersection "seam" that runs almost parallel to this torsional coordinate. At the lowest energy point on this conical intersection seam, the amino group is untwisted; however, the seam is accessible for a large range of torsional angles. Thus, the S(1) LE-TICT equilibration and dual fluorescence will be controlled by (a) the S(1) torsional reaction path and (b) the position along the amino group twist coordinate where the S(2)/S(1) CT-LE radiationless decay occurs. For DMABN, population of LE and TICT can occur because the two species have similar stabilities. However, in ABN, the equilibrium lies in favor of LE, as a TICT state was found at much higher energy with a low reaction barrier toward LE. This explains why dual fluorescence cannot be observed in ABN. The S(1)-->S(0) deactivation channel accessible from the LE state was also studied.

Journal Article↗

PubMed2004

Nuclear SDH2-1 and SDH2-2 genes, encoding the iron-sulfur subunit of mitochondrial complex II in Arabidopsis, have distinct cell-specific expression patterns and promoter activities.

Three different nuclear genes encode the essential iron-sulfur subunit of mitochondrial complex II (succinate dehydrogenase) in Arabidopsis (Arabidopsis thaliana), raising interesting questions about their origin and function. To find clues about their role, we have undertaken a detailed analysis of their expression. Two genes (SDH2-1 and SDH2-2) that likely arose via a relatively recent duplication event are expressed in all organs from adult plants, whereas transcripts from the third gene (SDH2-3) were not detected. The tissue- and cell-specific expression of SDH2-1 and SDH2-2 was investigated by in situ hybridization. In flowers, both genes are regulated in a similar way. Enhanced expression was observed in floral meristems and sex organ primordia at early stages of development. As flowers develop, SDH2-1 and SDH2-2 transcripts accumulate in anthers, particularly in the tapetum, pollen mother cells, and microspores, in agreement with an essential role of mitochondria during anther development. Interestingly, in contrast to the situation in flowers, only SDH2-2 appears to be expressed at a significant level in root tips. Strong labeling was observed in all cell layers of the root meristematic zone, and a cell-specific pattern of expression was found with increasing distance from the root tip, as cells attain their differentiated state. Analysis of transgenic Arabidopsis plants carrying SDH2-1 and SDH2-2 promoters fused to the beta-glucuronidase reporter gene indicate that both promoters have similar activities in flowers, driving enhanced expression in anthers and/or pollen, and that only the SDH2-2 promoter is active in root tips. These beta-glucuronidase staining patterns parallel those obtained by in situ hybridization, suggesting transcriptional regulation of these genes. Progressive deletions of the promoters identified regions important for SDH2-1 expression in anthers and/or pollen and for SDH2-2 expression in anthers and/or pollen and root tips. Interestingly, regions driving enhanced expression in anthers are differently located in the two promoters.

Arabidopsis↗

PubMed2004

Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate.

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.

Acrylamide↗

PubMed2004

Transfer of RPS14 and RPL5 from the mitochondrion to the nucleus in grasses.

Gene transfer from the mitochondrion to the nucleus, a process of outstanding importance to the evolution of the eukaryotic cell, is an on-going phenomenon in higher plants. After transfer, the mitochondrial gene has to be adapted to the nuclear context by acquiring a new promoter and targeting information to direct the protein back to the organelle. To better understand the strategies developed by higher plants to transfer organellar genes during evolution, we investigated the fate of the mitochondrial RPL5-RPS14 locus in grasses. While maize mitochondrial genome does not contain RPS14 and RPL5 genes, wheat mitochondrial DNA contains an intact RPL5 gene and a nonfunctional RPS14 pseudogene. RPL5 and PsiRPS14 are co-transcribed and their transcripts are edited. In wheat, the functional RPS14 gene is located in the nucleus, within the intron of the respiratory complex II iron-sulfur subunit gene (SDH2). Its organization and expression mechanisms are similar to those previously described in maize and rice, allowing us to conclude that RPS14 transfer and nuclear activation occurred before divergence of these grasses. Unexpectedly, we found evidence for a more recent RPL5 transfer to the nucleus in wheat. This nuclear wheat RPL5 acquired its targeting information by duplication of an existing targeting presequence for another mitochondrial protein, ribosomal protein L4. Thus, mitochondrial and nuclear functional RPL5 genes appear to be maintained in wheat, supporting the hypothesis that in an intermediate stage of the transfer process, both nuclear and mitochondrial functional genes coexist. Finally, we show that RPL5 has been independently transferred to the nucleus in the maize lineage and has acquired regulatory elements for its expression and a mitochondrial targeting peptide from an unknown source.

Amino Acid Sequence↗

PubMed2003

Molecular basis for Bacillus thuringiensis Cry1Ab toxin specificity: two structural determinants in the Manduca sexta Bt-R1 receptor interact with loops alpha-8 and 2 in domain II of Cy1Ab toxin.

The identification of epitopes involved in Cry toxin-receptor interaction could provide insights into the molecular basis of insect specificity and for designing new toxins to overcome the potential problem of insect resistance. In previous works, we determined that the Manduca sexta Cry1A cadherin-like receptor (Bt-R(1)) interacts with Cry1A toxins through epitope (865)NITIHITDTNN(875) and by loop 2 of domain II in the toxin (Gomez, I., Miranda-Rios, J., Rudiño-Piñera, E., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2002) J. Biol. Chem. 277, 30137-30143.). In this work, we narrowed to 12 amino acids a previously identified Bt-R(1) 66 amino acids epitope (Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S. A., Midbo, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Insect Biochem. Mol. Biol. 32, 1025-1036) and identified loop alpha-8 of Cry1Ab domain II as its cognate binding epitope. Two amino acid Bt-R(1) toxin binding regions of 70 residues, one comprised of residues 831-900 containing the (865)NITIHITDTNN(875) epitope (TBR1) and the other comprised of residues 1291-1360 (TBR2) were cloned by RT-PCR and produced in Escherichia coli. Cry1A toxins bind with the two TBR regions in contrast with the nontoxic Cry3A toxin. The loop 2 synthetic peptide competed with the binding of Cry1Ab toxin to both TBR regions in contrast to the alpha-8 synthetic peptide that only competed with Cry1Ab binding to TBR2. Western blots and competition ELISA analysis showed that the Cry1Ab loop 2 RR368-9EE mutant did not show observable binding to TBR1 but still bound the TBR2 peptide. This result suggests that loop alpha-8 interacts with the TBR2 region. Competition ELISA analysis of Cry1Ab binding to the two TBR peptides revealed that the toxin binds the TBR1 region with 6-fold higher affinity than the TBR2 region. The amino acid sequence of TBR2 involved on Cry1Ab interaction was narrowed to 12 amino acids, (1331)IPLPASILTVTV(1342), by using synthetic peptides as competitors for Cry1Ab binding to Bt-R(1). Our results show that the specificity of Cry1A involves at least two structural determinants on both molecules.

Animals↗

PubMed2003

[Importance of severe lesions left untreated in patients with acute coronary syndromes and angioplasty of the culprit lesion].

INTRODUCTION AND OBJECTIVES: Patients with acute coronary syndromes may have significantly stenotic nonculprit lesions that do not show complex lesion morphology. We investigated whether these lesions were prone to become unstable since they exist within a prothrombotic and inflammatory systemic milieu. PATIENTS AND METHOD: We evaluated the clinical course of 150 patients after successful angioplasty of a culprit lesion: 75 patients with a severely stenotic but uncomplicated nonculprit lesion (group A) and 75 patients without these lesions (group B). RESULTS: In group A, 1 patient (1.3%) required angioplasty of an initially nonculprit lesion, and in group B, 2 patients (2.6%) died in cardiogenic shock. After 1 year of follow-up, in group A, 4 patients (5.3%) died (cardiac deaths), 1 patient (1.3%) had a myocardial infarction, and 10 patients (13.3%) underwent a repeat revascularization procedure, which in 6 cases (8%) was angioplasty of an initially nonculprit lesion. In all 6 patients with angioplasty of the initially nonculprit lesion, revascularization was done within the first 4 months and was indicated for unstable angina. In group B, 1 patient (1.3%) died (noncardiac death) and 2 patients (2.6%) underwent a repeat revascularization procedure because of restenosis. Survival curves were significantly different between both groups. Belonging to group A was the only independent predictor for events, and within this group location of the lesion in the left anterior descending artery was the main predictor. CONCLUSIONS: The presence of nonculprit lesions of uncomplicated morphology at the time of a percutaneous revascularization procedure for a culprit lesion in patients with acute coronary syndrome is a short- and middle-term predictor of a moderate rate of recurrent events when these initially innocuous lesions become unstable.

Acute Disease↗

PubMed2002

Thermal and photochemical rearrangement of bicyclo[3.1.0]hex-3-en-2-one to the ketonic tautomer of phenol. Computational evidence for the formation of a diradical rather than a zwitterionic intermediate.

The ground state (S(0)) and lowest-energy triplet state (T(1)) potential energy surfaces (PESs) concerning the thermal and photochemical rearrangement of bicyclo[3.1.0]hex-3-en-2-one (8) to the ketonic tautomer of phenol (11) have been extensively explored using ab initio CASSCF and CASPT2 calculations with several basis sets. State T(1) is predicted to be a triplet pipi lying 66.5 kcal/mol above the energy of the S(0) state. On the S(0) PES, the rearrangement of 8 to 11 is predicted to occur via a two-step mechanism where the internal cyclopropane C-C bond is broken first through a high energy transition structure (TS1-S(0)()), leading to a singlet intermediate (10-S(0)()) lying 25.0 kcal/mol above the ground state of 8. Subsequently, this intermediate undergoes a 1,2-hydrogen shift to yield 11 by surmounting an energy barrier of only 2.7 kcal/mol at 0 K. The rate-determining step of the global rearrangement is the opening of the three-membered ring in 8, which involves an energy barrier of 41.2 kcal/mol at 0 K. This high energy barrier is consistent with the fact that the thermal rearrangement of umbellulone to thymol is carried out by heating at 280 degrees C. Regarding the photochemical rearangement, our results suggest that the most efficient route from the T(1) state of 8 to ground state 11 is the essentially barrierless cleavage of the internal cyclopropane C-C bond followed by radiationless decay to the S(0) state PES via intersystem crossing (ISC) at a crossing point (S(0)()/T(1)()-1) located at almost the same geometry as TS1-S(0)(), leading to the formation of 10-S(0)() and the subsequent low-barrier 1,2-hydrogen shift. The computed small spin-orbit coupling between the T(1) and S(0) PESs at S(0)()/T(1)()-1 (1.2 cm(-)(1)) suggests that the ISC between these PESs is the rate-determining step of the photochemical rearrangement 8 --> 11. Finally, computational evidence indicates that singlet intermediate 10-S(0)() should not be drawn as a zwitterion, but rather as a diradical having a polarized C=O bond.

Journal Article↗

PubMed2002

Pore formation activity of Cry1Ab toxin from Bacillus thuringiensis in an improved membrane vesicle preparation from Manduca sexta midgut cell microvilli.

The pore formation activity of Cry1Ab toxin is analyzed in an improved membrane preparation from apical microvilli structures of Manduca sexta midgut epithelium cells (MEC). A novel methodology is described to isolate MEC and brush border membrane vesicles (BBMV) from purified microvilli structures. The specific enrichment of apical membrane enzyme markers aminopeptidase (APN) and alkaline phosphatase (APh) were 35- and 22-fold, respectively, as compared to the whole midgut cell homogenate. Ligand-blot and Western-blot experiments showed that Cry1A specific receptors were also enriched. The pore formation activity of Cry1Ab toxin was fourfold higher in the microvilli membrane fraction that showed low intrinsic K+ channels and higher APN and APh activities than in the basal-lateral membrane fraction harboring high intrinsic K+ channels. These data suggest that basal-lateral membrane was separated from apical membrane.This procedure should allow more precise studies of the interaction of Cry toxins with their target membranes, avoiding unspecific interaction with other cellular membranes, as well as the study of the pore formation activity induced by Cry toxins in the absence of endogenous channels from M. sexta midgut cells.

Alkaline Phosphatase↗

PubMed2002

Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin.

Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin-like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore-forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix alpha-1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8-anilino-1-naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane-associated protease. Finally, a model where receptor binding allows the efficient cleavage of alpha-1 and formation of a pre-pore oligomeric structure that is efficient in pore formation, is presented.

Bacillus thuringiensis↗

PubMed2002

Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation.

Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation.

Animals↗