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Biomedical subjects

A Bravo

Publications and source records attributed to A Bravo.

At least 19 recordsLinked to original sources
PubMed2007

Permeability changes of Manduca sexta midgut brush border membranes induced by oligomeric structures of different cry toxins.

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K(+) permeability. A fluorometric assay performed with 3,3'-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K(+)-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

Animals↗
PubMed2006

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.

Bacillus thuringiensis↗
PubMed2005

Skin immune responses in cattle after primary and secondary infections with Hypoderma lineatum (Diptera: Oestridae) larvae.

Cellular immune responses were examined in the skin of cattle after primary and secondary experimental infections with Hypoderma lineatum larvae. Skin biopsies were taken at 0, 6, 12, 48 and 96 h post-infection (h.p.i.). In primary infected animals the penetration of Hypoderma larvae was characterized by moderate inflammatory responses. The pattern of cellular changes in previously infected animals suggested an allergic or rheumatic process, probably as a consequence of the development of a type III hypersensitivity reaction against the second or successive reinfections. Perivascular infiltration with CD3(+) T lymphocytes was marked in infected groups, but especially in previously infected animals, with a significant increase with respect to uninfected controls at 48 h.p.i. B cells remained close to control values during primary infection, while they increased significantly 12 h.p.i. in reinfected animals. IgG(+) plasma cells were also very abundant during secondary infections, with significant differences from primary infected and uninfected animals from 6 h.p.i. onwards. These results suggest that secondary infections allow cellular responses which may be effective in killing some of the entering larvae, resulting in a degree of resistance.

Animals↗
PubMed2005

Oxidative status during late pregnancy and early lactation in dairy cows.

In the last few years, the detection of free radical damage and the body's defences against it have become increasingly important in clinical medicine as a complementary tool in the evaluation of metabolic status. The aim of this study was to evaluate, under field conditions, the anti-oxidant status of healthy cows during late pregnancy and lactation onset using two parameters: (1) plasma levels of malondialdehyde (MDA), a degradation product of lipid peroxidation, and (2) total antioxidant status (TAS). Results were compared with those obtained in another group of cows with lesser metabolic demands. We also investigated possible relationships between antioxidant status markers and other relevant blood parameters. Our results confirmed the characteristic metabolic changes associated with late pregnancy and early lactation. MDA and TAS provided an accurate reflection of the internal physiological status of the animal. The data indicated increased lipid peroxidation around parturition, but with wide individual variations that may be attributable not only to the physiological stage but also to unknown factors that will have to be further considered in future studies.

Animals↗
PubMed2005

[Improved short-term cardiovascular profile after simultaneous pancreas-kidney transplantation].

The prognosis of type 1 diabetes mellitus (T1DM) patients with chronic renal failure (CRF) improves after simultaneous pancreas-kidney (SPK) transplantation. In order to evaluate the changes in cardio-vascular risk (CVR) factors after SKP, we studied nine recipients before and 6 months after SPK. There were five females and four males, with a mean age of 37 +/- 8 years, duration of diabetes of 24 +/- 5 years, three of them before starting dialysis, and six on dialysis (hemodialysis = 5; peritoneal dialysis = 1). Before SPK, all patients received anti-hypertensive therapy (1-4 drugs; mean 2.2 +/- 0.9) and eight received statins. At 6 months after SPK, all patients were under triple immunosuppressive therapy (steroids + tacrolimus + MMF) without statins. They had normal renal function (Plasma Creatinine = 1.2 +/- 0.3 mg/dl) and pancreatic endocrine function (glycemia = 80 +/- 8 mg/dl). HbA1c decreased significantly (8.4 +/- 1.2 vs 4.7 +/- 0.6%; p < 0.007) with a value > 7% in seven patients before SPK and in none 6 months after SKP transplantation (p < 0.001). Although Body Mass Index increased (23 +/- 2 vs 25 +/- 3 kg/m2; p < 0.05), plasma triglycerides decreased (130 +/- 51 vs 88 +/- 33 mg/dl; p < 0.05), and total cholesterol, LDL-cholesterol and HDL-cholesterol were similar. Systolic and diastolic blood pressure (BP) decreased (156 +/- 7 vs 133 +/- 15; p < 0.01 and 96 +/- 7 vs 79 +/- 9; p < 0.007) with only two patients on anti-hypertensive therapy (1 drug). Likewise, before transplantation all patients were hypertensive (six grade 1 and three grade 2) while this was observed in only two at the end of follow-up (both grade 1) (p < 0.001). In conclusion, SPK transplantation with good renal and pancreatic function is associated with a short-term improvement in CVR profile.

Adult↗
PubMed2004

Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains.

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.

Animals↗
PubMed2004

Role of toxin activation on binding and pore formation activity of the Bacillus thuringiensis Cry3 toxins in membranes of Leptinotarsa decemlineata (Say).

Binding and pore formation constitute key steps in the mode of action of Bacillus thuringiensis delta-endotoxins. In this work, we present a comparative analysis of toxin-binding capacities of proteolytically processed Cry3A, Cry3B and Cry3C toxins to brush border membranes (BBMV) of the Colorado potato beetle Leptinotarsa decemlineata (CPB), a major potato coleopteran-insect pest. Competition experiments showed that the three Cry3 proteolytically activated toxins share a common binding site. Also heterologous competition experiments showed that Cry3Aa and Cry3Ca toxins have an extra binding site that is not shared with Cry3Ba toxin. The pore formation activity of the three different Cry3 toxins is analysed. High pore-formation activities were observed in Cry3 toxins obtained by proteolytical activation with CPB BBMV in contrast to toxins activated with either trypsin or chymotrypsin proteases. The pore-formation activity correlated with the formation of soluble oligomeric structures. Our data support that, similarly to the Cry1A toxins, the Cry3 oligomer is formed after receptor binding and before membrane insertion, forming a pre-pore structure that is insertion-competent.

Animals↗
PubMed2004

Activity of Lutzomyia pseudolongipalpis and L. longipalpis s.l. (Diptera: Psychodidae) in Venezuela.

The nocturnal activity of the phlebotomine sandfly Lutzomyia pseudolongipalpis and two populations of L. longipalpis s.l. from different American visceral leishmaniasis foci in Venezuela was studied using collection bottle rotator traps. The activity of L. pseudolongipalpis from Lara State was continuous throughout the night, while that of L. longipalpis s.l. from El Layero, Guárico State and from Santa Ana del Valle, Margarita Island, was greatest before 23:00 h. The activity of sandflies of both populations and sexes steadily decreased thereafter. These different patterns seem to correlate with genetic data that indicate the presence in Venezuela of at least two sibling species in the L. longipalpis complex. The advantages of the bottle rotator trap for this type of study are discussed.

Animals↗
PubMed2003

Effect of ursodeoxycholic acid on the impairment induced by maternal cholestasis in the rat placenta-maternal liver tandem excretory pathway.

We investigated the effects of ursodeoxycholic acid (UDCA; 60 microg/day/100 g b.wt.) on the impairment induced by maternal obstructive cholestasis during pregnancy (OCP) in the rat placenta-maternal liver tandem excretory pathway. A blunted catheter was implanted in the common bile duct on day 14 of pregnancy, and the tip was cut on day 21. [(14)C]Glycocholate (GC) was then administered through the umbilical artery of "in situ" perfused placenta (placental transfer test) or through the maternal jugular vein (biliary secretion test), and GC bile output was measured. OCP impaired both GC placental transfer and maternal biliary secretion. UDCA moderately improved the latter but had a more marked beneficial effect on GC placental transfer. Histological examination revealed trophoblast atrophy and structural alterations, e.g., loss of apical membrane microvilli in OCP placentas. Gene expression level was investigated by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. OCP reduced both placental lactogen II (a trophoblast-specific gene) mRNA and the functional amount of epithelial tissue, determined by transplacental diffusion of antipyrin. Using a rapid filtration technique, impairment in the ATP-dependent GC transport across trophoblast apical plasma membranes obtained from OCP placentas was found. UDCA partially prevented all these changes. The expression level of organic anion transporters Oatp1, Oatp2, and Oatp4, and multidrug resistance-associated proteins Mrp1, Mrp2, and Mrp3 in whole placenta were not affected or were moderately affected by OCP but greatly enhanced by UDCA. In summary, UDCA partially prevents deleterious effects of OCP on the rat placenta-maternal liver tandem excretory pathway, mainly by preserving trophoblast structure and function.

Animals↗
PubMed2001

Self-affine nature of the stress-strain behavior of thin fiber networks.

The stress-strain behavior of toilet paper is studied. We find that the damaged parts of stress-strain curves possess a self-affine scaling invariance. Moreover, we find that the stress-strain behavior and the rupture line roughness are characterized by the same scaling (Hurst) exponent H, which is not universal: rather it changes from sample to sample. The variations on H are mainly due to fluctuations in the paper structure, which are larger than statistical errors within a sample. Furthermore, the same exponent governs the changes in the stress-strain curve as the strain rate increases. The fractal damage model is employed to explain experimental observations.

Journal Article↗
PubMed2001

Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation.

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60-65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices alpha1 and alpha2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the alpha1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.

Animals↗
PubMed2001

Mode of action of Bacillus thuringiensis PS86Q3 strain in hymenopteran forest pests.

The mode of action of Cry toxins has been described principally in lepidopteran insects as a multistep process. In this work we describe the mode of action of a Cry toxin active in the common pine sawfly Diprion pini (Hymenoptera, Diprionidae), considered a major forest pest in Europe. Strain PS86Q3 contains a long bipyramidal crystal composed of five major proteins. The N-terminal sequence shows that the 155 kDa protein corresponds to Cry5B toxin and the other proteins belong to the Cry5A subgroup. PCR analysis indicates the presence of cry5Ac and cry5Ba genes, suggesting that Cry5A protein should be Cry5Ac. Activation of protoxins with trypsin or with midgut content from D. pini and Cephacia abietis (Hymenoptera, Pamphiliidae) (spruce webspinning sawfly), another important hymenopteran forest pest, produced a single 75 kDa toxin that corresponded to Cry5A by N-terminal sequence and is responsible for the insecticidal activity. Homologous competition experiments with D. pini and C. abietis brush border membrane vesicles (BBMV) showed that the binding interaction of Cry5A is specific. Membrane potential measurements using a fluorescent dye indicate that Cry5A toxin at nM concentration caused immediate permeability changes in the BBMV isolated from both hymenopteran larvae. The initial response and the sustained permeability change are cationic as previously shown for Cry1 toxins. These results indicate that the hymenopteran specific Cry5A toxin exerts toxicity by a similar mechanism as Cry1 toxins.

Animals↗
PubMed2001

Altered skin development and impaired proliferative and inflammatory responses in transgenic mice overexpressing the glucocorticoid receptor.

Glucocorticoids (GCs) are potent inhibitors of epidermal proliferation and effective anti-inflammatory compounds, which make them the drug of choice for a wide range of inflammatory and hyperproliferative skin disorders. GC action is mediated via the glucocorticoid receptor (GR). To study the role of GR in skin development and the molecular mechanisms underlying its action, we generated transgenic mice overexpressing GR in epidermis and other stratified epithelia, under the control of the keratin K5 promoter. Newborn mice show altered skin development, manifested as variable-sized skin lesions that range from epidermal hypoplasia and underdeveloped dysplastic hair follicles to a complete absence of this tissue. In the most affected individuals, skin was absent at the cranial and umbilical regions, and the vibrissae and eyebrows appear scarce, short, and curly. In addition, as a consequence of transgene expression in other ectodermally derived epithelia, K5-GR mice exhibited further abnormalities that strikingly resemble the clinical findings in patients with ectodermal dysplasia, which includes aplasia cutis congenita. In adult transgenic skin, topical application of the tumor promoter TPA did not elicit hyperplasia or transcriptional induction of several proinflammatory cytokines. This anti-inflammatory role of GR was due at least in part to interference with NF-kB, leading to a strong reduction in the kB-binding activity without altering the transcriptional levels of the inhibitor IkBa.

Animals↗
PubMed2001

Mapping the epitope in cadherin-like receptors involved in Bacillus thuringiensis Cry1A toxin interaction using phage display.

In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K(d)) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20-51 nm. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R(1)), Bombyx mori (Bt-R(175)), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R(1) or Bt-R(175) are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R(1) or Bt-R(175) receptors. Finally, we showed that synthetic peptides homologous to Bt-R(1) and scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R(1) and Bt-R(175) involved in Cry1A toxin interaction.

Amino Acid Sequence↗
PubMed2001

A single amino acid substitution within a coiled-coil motif changes the assembly of a 53-amino acid protein from two-dimensional sheets to filamentous structures.

The bacteriophage phi29 replication protein p1 self-interacts in vitro, generating highly ordered structures. Specifically, the 53-amino acid protein p1DeltaN33, which retains the sequence of p1 spanning amino acids Met(34) to Lys(85), assembles into two-dimensional protofilament sheets. The region of protein p1 located between residues Glu(38) and Asn(65) presumably forms an alpha-helical coiled-coil structure. Here we have examined the role of this coiled-coil sequence in the formation of protofilament sheets. Using sedimentation assays and negative-stain electron microscopy analysis, we demonstrate that residues Leu(46), Met(53), and Leu(60), but not Leu(39), are essential for p1DeltaN33 assembly into sheets. Remarkably, replacement of Leu(46) by Val shifts the pathway of molecular assembly, leading to the formation of filamentous polymers approximately 10 nm in diameter. These results show, for the first time, that a short coiled-coil motif can mediate protein assembly into protofilament sheet structures.

Amino Acid Sequence↗
PubMed2001

Structural and functional studies of alpha-helix 5 region from Bacillus thuringiensis Cry1Ab delta-endotoxin.

The crystal insecticidal proteins from Bacillus thuringiensis are modular proteins comprised of three domains connected by single linkers. Domain I is a seven alpha-helix bundle, which has been involved in membrane insertion and pore formation activity. Site-directed mutagenesis has contributed to identify regions that might play an important role in the structure of the pore-forming domain within the membrane. There are several evidences that support that the hairpin alpha4-alpha5 inserts into the membrane in an antiparallel manner, while other helices lie on the membrane surface. We hypothesized that highly conserved residues of alpha5 could play an important role in toxin insertion, oligomerization and/or pore formation. A total of 15 Cry1Ab mutants located in six conserved residues of Cry1Ab, Y153, Y161, H168, R173, W182 and G183, were isolated. Eleven mutants were located within helix alpha5, one mutant was located in the loop alpha4-alpha5 and three mutants, W182P, W182I and G183C, were located in the loop alpha5-alpha6. Their effect on binding, K(+) permeability and toxicity against Manduca sexta larvae was analyzed and compared. The results provide direct evidence that some residues located within alpha5 have an important role in stability of the toxin within the insect gut, while some others also have an important role in pore formation. The results also provide evidence that conserved residues within helix alpha5 are not involved in oligomer formation since mutations in these residues are able to make pores in vitro.

Amino Acid Sequence↗