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Biomedical subjects

A Bravo

Publications and source records attributed to A Bravo.

At least 37 records · Page 2Linked to original sources
PubMed2001

How Bacillus thuringiensis has evolved specific toxins to colonize the insect world.

Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. Together the subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. This is mainly determined by the arsenal of crystal proteins that the bacterium produces during sporulation. Here we describe the properties of these toxin proteins and the current knowledge of the basis for their specificity. Assessment of phylogenetic relationships of the three domains of the active toxin and experimental results indicate how sequence divergence in combination with domain swapping by homologous recombination might have caused this extensive range of specificities.

Animals↗
PubMed2001

Inflammatory changes after cryosurgery-induced necrosis in human melanoma xenografted in nude mice.

There is growing evidence that necrosis, instead of apoptosis, could act as a natural adjuvant, which could activate an immune response. In this work we have investigated if induction of tumor necrosis could trigger the affluence of inflammatory cells at the tumor site, and thus induce an immune response. For this purpose, a liquid N2 spray was applied on human melanoma (IIB-MEL-J cell line) xenografted in nude mice and 24 h later some mice received intratumorally a single 500 U dose of recombinant murine granulocyte macrophage-colony-stimulating factor. 77-100% of the tumor mass underwent necrosis. Congestion, edema, and endothelial cell activation were the first noticeable events. A quick infiltrative response of polymorphonuclear leukocytes around the tumor was detected 24 h after liquid N2 application, peaking at day 3. Massive macrophage recruitment was observed since day 3. An early intratumoral infiltration with inflammatory cells was only detected in the group that received recombinant murine granulocyte macrophage- colony-stimulating factor after necrosis induction by liquid N2. Coexisting DEC 205- and F4/80-positive cells increased in number, and their localization was predominantly peritumoral after necrosis. Antibody response was only detected in the groups with tumor-induced necrosis. Our results suggest that cryosurgery-induced necrosis could be a useful model to analyze the interaction among necrosis, inflammation, and the generation of an immune response.

Animals↗
PubMed2001

Recurrent pneumonia as warning manifestation for suspecting primary immunodeficiencies in children.

Two hundred and eight children with recurrent pneumonia were studied over a 5-year period. Among these patients we found 10 cases with primary immunodeficiency disease: 6 cases of IgA deficiency, 1 case of X-linked agammaglobulinemia, 1 case of common variable immunodeficiency, 1 case of hyper IgM syndrome, and 1 case of Wiskott-Aldrich syndrome. This study describes the clinical features of these cases and assesses the usefulness of our immunodeficiency screening protocol. In this group 6 were males; the mean age at first episode of pneumonia was 3 years (range 3 months to 18 years), and the age of diagnosis ranged between 10 months and 19 years. The average number of episodes of pneumonia in each patient was 5 (range 2 to 12), and the number of hospitalizations ranged up to 13. The etiologic agents isolated from this recurrent pneumonia were S. pneumoniae, Moraxella, adenovirus, respiratory syncytial virus, and influenza B virus. Intravenous immunoglobulin was used in four cases. Two patients had chronic pulmonary damage with bronchiectasis and interstitial pneumonia. Only one patient died (Wiskott-Aldrich syndrome) during the follow-up from an intracranial hemorrhage. We found that the screening protocol applied to patients with recurrent pneumonia is a useful tool for ruling out the primary immunodeficiency disorders.

Adolescent↗
PubMed2000

Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells.

Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3-5 x 10(6) interleukin 12 (IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5-1 x 10(6) CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed, regardless of the amount of injected CT26-IL12 cells. On the contrary, a strong increase in anti-CT26-specific IgG2a levels was observed only when 3-5 x 10(6) CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3-5 x 10(6) CT26-IL12 cells developed local nodules for a few days, which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+ T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.

Animals↗
PubMed2000

Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1.

The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.

Adenosine Monophosphate↗
PubMed2000

Evidence for intermolecular interaction as a necessary step for pore-formation activity and toxicity of Bacillus thuringiensis Cry1Ab toxin.

Based on the observation of large conductance states formed by Bacillus thuringiensis Cry toxins in synthetic planar lipid bilayers and the estimation of a pore size of 10-20 A, it has been proposed that the pore could be formed by an oligomer containing four to six Cry toxin monomers. However, there is a lack of information regarding the insertion of Cry toxins into the membrane and oligomer formation. Here we provide direct evidence showing that the intermolecular interaction between Cry1Ab toxin monomers is a necessary step for pore formation and toxicity. Two Cry1Ab mutant proteins affected in different steps of their mode of action (F371A in receptor binding and H168F in pore formation) were affected in toxicity against Manduca sexta larvae. Binding analysis showed that F371A protein bound more efficiently to M. sexta brush border membrane vesicles when mixed with H168F in a one to one ratio. These mutant proteins also recovered pore-formation activity, measured with a fluorescent dye with isolated brush border membrane vesicles, and toxicity against M. sexta larvae when mixed, showing that monomers affected in different steps of their mode of action can form functional hetero-oligomers.

Animals↗
PubMed2000

Effect of maternal cholestasis on bile acid transfer across the rat placenta-maternal liver tandem.

Cholestasis of pregnancy induces alterations in bile acid transport by human trophoblast plasma membrane (TPM) vesicles. We investigated whether maternal cholestasis affects the overall ability of the rat placenta to carry out vectorial bile acid transfer from the fetus to the mother. Complete obstructive cholestasis (OCP) was maintained during the last week of pregnancy and released at term (day 21), before experiments were performed. In situ single-pass perfusion of one placenta per rat with 250 nmol [(14)C]glycocholic acid (GC) revealed an impaired uptake in OCP rats (2.28 vs. 5.53 nmol in control rats). Approximately 100% of GC taken up by control placentas was secreted in maternal bile over 120 minutes (5.38 nmol), whereas this was only 61% (1.40 nmol) of the GC taken up by OCP placentas. When 5 nmol GC was administered through the jugular vein no significant difference between both groups in total GC bile output was found. The efficiency (V(max)/K(M)) of adenosine triphosphate (ATP)-dependent GC transport by vesicles from the maternal side of TPM was decreased (-41%) in OCP. Moreover, histological examination of the placentas suggested a reduction in the amount of functional trophoblast in the OCP group. This was consistent with a lower antipyrine diffusion across the placenta in these animals. In sum, our results indicate that maternal cholestasis affects the ability of the placenta to efficiently carry out bile acid transfer from fetal to maternal blood. Changes in both the structure and the functionality of the chorionic tissue may account for this impairment.

Animals↗
PubMed1999

The oligomeric state of Bacillus thuringiensis Cry toxins in solution.

The molecular mass of different Cry toxins produced by Bacillus thuringiensis bacteria was estimated by size-exclusion chromatography and non-denaturing polyacrylamide gel electrophoresis at neutral and alkaline pH in order to assess the existence of oligomers in solution. We found that Cry1Aa, Cry1Ac, Cry1C, Cry1D and Cry3A toxins exist in solution as a mixture of monomer and high molecular mass aggregates with an apparent molecular mass greater than 600 kDa, that depend on the time elapsed between toxin activation and analysis. Aggregation of toxins by disulfide bonds is unlikely because aggregates are also observed in samples incubated with DTT. These data show that the Cry toxins studied do not form oligomers of less than ten subunits in solution and suggest that oligomer formation may occur after the toxin binds to the receptor and inserts into the membrane.

Bacillus thuringiensis↗
PubMed1999

IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response.

In spite of the evidence that IL-10 has Th1-immunosuppressive and anti-inflammatory effects, it has been shown that IL-10 may reduce the tumorigenic capacity of certain tumor cell types. In order to characterize the responses elicited by IL-10, we explored the effect of transducing murine CT26 colon carcinoma cells with a recombinant retrovirus expressing mIL-10. IL-10 gene transfer of CT26 cells had no effect on tumor cell growth on plastic surface but inhibited the anchorage-independent growth capacity of tumor cells and their metastatic potential as assessed by their invasive and migration ability. Expression of IL-10 also elicited an antitumor immune response involving both CD4+ and CD8+ T cells. Assessment of the immune status of the animals demonstrated that mice injected with CT26-IL10 cells showed prevalence of a systemic and tumor-specific Th2 response. Spleen cells obtained from these mice showed an increased production of IL-4 and no changes in IFNgamma levels, characteristic of a Th2 response. These results demonstrate that IL-10 affects CT26 tumor cell growth by both inhibiting the malignant phenotype and by recruiting and activating a T cell-mediated antitumor response. This T cell response occurs in the context of a shift towards a Th2 response.

Analysis of Variance↗
PubMed1999

Exocrine pancreatic disorders in transsgenic mice expressing human keratin 8.

Keratins K8 and K18 are the major components of the intermediate-filament cytoskeleton of simple epithelia. Increased levels of these keratins have been correlated with various tumor cell characteristics, including progression to malignancy, invasive behavior, and drug sensitivity, although a role for K8/K18 in tumorigenesis has not yet been demonstrated. To examine the function of these keratins, we generated mice expressing the human K8 (hk8) gene, which leads to a moderate keratin-content increase in their simple epithelia. These mice displayed progressive exocrine pancreas alterations, including dysplasia and loss of acinar architecture, redifferentiation of acinar to ductal cells, inflammation, fibrosis, and substitution of exocrine by adipose tissue, as well as increased cell proliferation and apoptosis. Histological changes were not observed in other simple epithelia, such as the liver. Electron microscopy showed that transgenic acinar cells have keratins organized in abundant filament bundles dispersed throughout the cytoplasm, in contrast to control acinar cells, which have scarce and apically concentrated filaments. The phenotype found was very similar to that reported for transgenic mice expressing a dominant-negative mutant TGF-beta type II receptor (TGFbetaRII mice). We show that these TGFbetaRII mutant mice also have elevated K8/K18 levels. These results indicate that simple epithelial keratins play a relevant role in the regulation of exocrine pancreas homeostasis and support the idea that disruption of mechanisms that normally regulate keratin expression in vivo could be related to inflammatory and neoplastic pancreatic disorders.

Animals↗
PubMed1998

Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets.

Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.

Amino Acid Sequence↗
PubMed1998

Analysis of sequences controlling tissue-specific and hyperproliferation-related keratin 6 gene expression in transgenic mice.

Keratin 6 (K6) is an intermediate filament protein found in hair follicles and in several internal stratified epithelia. This keratin has been the focus of special attention because it is also strongly induced in epidermal interfollicular keratinocytes in hyperproliferative situations and in certain conditions leading to abnormal differentiation. To localize and identify the sequences controlling this complex expression pattern, and because of their potential use in transgenic mouse models and gene therapy strategies for epidermal hyperproliferative disorders, we have thoroughly analyzed a 9 kbp region of this gene previously shown to direct proper tissue-specific and inducible expression in transgenic mice. To reproduce the K6 constitutive expression pattern, cooperation is necessary between elements located in at least two different regions, one distal between -9 and -4 kbp and one proximal between -830 and -125 bp with respect to the CAP site. The ability to induce expression under hyperproliferative conditions resides in the 2.4 kbp fragment preceding the transcription start site. When this DNA fragment was analyzed in more detail, we found that all subfragments tested contained regulatory elements necessary for inducible expression. Thus, a complex organization of K6 regulatory elements emerges, as both the constitutive and the inducible expressions of this gene are under the control of multiple elements dispersed throughout relatively large 5' flanking DNA fragments. These findings will allow the expression of cloned genes in transgenic mouse skin in response to pathological or applied hyperproliferative stimuli, avoiding the effects of their constitutive expression in other epithelia.

Animals↗
PubMed1998

Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection.

Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity. A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country. The characterization of the strain collection provided useful information on the ecological patterns of distribution of B. thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products. The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes. The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects. The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects. The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal. The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects. Six pairs of general primers are used in this method. Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers. Strains containing cry1 genes were the most abundant in our collection (49.5%). Thirty-three different cry1-type profiles were identified. B. thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes. cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively. No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found. Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera. Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.

Animals↗
PubMed1998

[Antitumor gene therapy using suicide genes].

Tumor cells transduced with retrovirus carrying the herpes simplex-1 virus thymidine kinase (HSV-tk) are capable of transforming the antiviral drug ganciclovir (GVC) into a metabolic form only toxic to dividing cells. The efficiency of this suicide gene therapy is increased by a "bystander" effect resulting not only in the death of the recipient cell, but also in the death of non modified surrounding cells. Even though the mechanism of this "bystander" effect remains to be elucidated, strong evidence suggest that the immune system plays a main role to achieve complete tumor eradication. In the present study we evaluate the efficiency of this suicide system on three different tumor models: one human melanoma, one murine melanoma, and a rat glioblastoma. Tumors were established by injection of tumor cells s.c. in nude and C57Bl/6 mice, respectively, and stereotactically into the brain of Sprague Dawley rats. Animals in the treated group were co-injected with packaging cells producing recombinant retrovirus carrying the HSV-tk gene, and followed by i.p. administration of GVC. In short term studies, we observed inhibition of tumor growth for all the tumor models evaluated (p < 0.01). In long term studies, using the C6 rat glioma line, 50% of the animals survived longer than 75 days (p < 0.0001), and were able to reject a contralateral challenges with C6 parental cells. Histological and immunohistochemical analysis showed the presence at an inflammatory infiltrate composed by T lymphocytes, macrophages and polymorphonuclear cells. These data demonstrate that suicide genes might represent an attractive form of cancer gene therapy in the treatment of brain tumors and their intracerebral dissemination.

Animals↗
PubMed1997

Aminopeptidase dependent pore formation of Bacillus thuringiensis Cry1Ac toxin on Trichoplusia ni membranes.

The insecticidal Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds to a 120 kDa aminopeptidase N (APN) in the midgut of susceptible insects such as Manduca sexta, Heliothis virescens, Lymantria dispar and Plutella xylostella. The 120 kDa APN has a glycosylphosphatidylinositol (GPI) anchor susceptible to the action of GPI-specific phospholipase C (PIPLC). Here we show that Cry1Ac pore-forming activity depends on the amount of APN present on brush border membrane vesicles (BBMV) from Trichoplusia ni larvae. Inhibition of APN activity with bestatin did not affect Cry1Ac pore formation, suggesting that Cry1Ac action depends on the presence of APN, but not on its enzymatic activity. N-Acetyl-D-galactosamine blocks the action of the toxin, indicating that this sugar is also directly involved in the Cry1Ac toxin-receptor interaction. Membrane potential measurements using PIPLC treated and non-treated BBMV suggest that both APN could participate as Cry1Ac receptor. The kinetic characterization of PIPLC sensitive and resistant APN indicates that they could be different isoforms. Finally, we show that in the presence of 200 mM Cs+ intrinsic BBMV T. ni channel permeability is not observed, while the toxin induced permeability is not affected, allowing an accurate analysis of the effect of the Cry1Ac toxin on T. ni midgut membranes.

Acetylgalactosamine↗
PubMed1997

Isolated domain II and III from the Bacillus thuringiensis Cry1Ab delta-endotoxin binds to lepidopteran midgut membranes.

The DNA fragment encoding Cry1Ab domain II-III (45.3 kDa) was cloned and expressed. Domain II-III is expressed in low yields. In vitro binding analysis to Manduca sexta and Trichoplusia ni larval midgut tissue sections demonstrated that domain II-III fragment bound along the microvilli of the midgut epithelium, indicating that this fragment retains binding functionality in the absence of domain I. Binding of domain II-III to the midgut brush border membrane proteins from T. ni larvae indicated that Cry1Ab toxin and domain II-III bind to the same 150 kDa protein. In contrast, in M. sexta membranes, Cry1Ab toxin binds to 200 and 120 kDa proteins, and domain II-III only binds to the 200 kDa protein. Finally, binding assays with isolated brush border membrane vesicles showed that the interaction of domain II-III with the membrane vesicles is highly reversible, supporting the proposition that the integration of domain I into the membrane could participate in the irreversible binding of the toxin. These studies confirm that this part of the toxin is involved in binding interactions and could be separated as a discrete fragment that conserves at least part of its functionality.

Animals↗
PubMed1997

Initiation of bacteriophage phi29 DNA replication in vivo: assembly of a membrane-associated multiprotein complex.

Initiation of in vitro phage phi29 DNA replication requires the formation of a heterodimer between a free molecule of terminal protein (TP), which acts as primer, and the viral DNA polymerase. We have analyzed membrane vesicles from phi29-infected Bacillus subtilis cells by quantitative immunoblot techniques. During phage DNA synthesis, large amounts of the viral proteins p1 and free TP were recovered in membrane fractions, as well as a low percentage of the total viral DNA polymerase. Interestingly, the amount of DNA polymerase in membrane fractions increased when viral DNA replication was blocked. Both protein p1 and free TP showed affinity for membranes in the absence of viral DNA. The association of protein p1 with membranes was abolished when the C-terminal 43 amino acid residues were deleted. The above results, together with the critical role of protein p1 for in vivo phi29 DNA replication, led us to conclude that a preliminary stage in the initiation of in vivo phi29 DNA replication could be the assembly of a membrane-associated multiprotein complex containing at least protein p1, free TP and DNA polymerase. Membrane-attachment of this complex could be directly mediated by both protein p1 and free TP. The ability of free TP to bind to membranes and to prime phi29 DNA replication would enable a nascent viral DNA molecule to become membrane-associated when its synthesis begins. We postulate that a general function of the TPs covalently linked to linear DNA genomes in prokaryotes might be, in addition to act as primer, to anchor the linear DNA molecule to the bacterial membrane.

Bacillus Phages↗