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Biomedical subjects

A Bravo

Publications and source records attributed to A Bravo.

At least 55 records · Page 3Linked to original sources
PubMed1997

A medical school's plan for anti-tobacco-use education in community schools and hospitals.

BACKGROUND: Tobacco use in the United States remains a significant health concern. In 1992, students at Dartmouth Medical School began a community service program that targeted the prevention of local tobacco use. METHODS: The program combined primary prevention, through education of school-aged children, with secondary prevention, through smoking cessation clinics. RESULTS: The program has received widespread community as well as national support for its actions. The program is in its initial stages of gathering data to determine whether there has been a significant impact on the tobacco use of the community. CONCLUSIONS: This paper is an introduction to the program's efforts, and a potential template for other medical schools to follow.

Adolescent↗
PubMed1997

Susceptibility of Lutzomyia longipalpis (Diptera: Psychodidae) to selected insecticides in an endemic focus of visceral leishmaniasis in Venezuela.

A field population of Lutzomyia longipalpis from La Rinconada, Lara State, an endemic focus of visceral leishmaniasis in Venezuela, was tested for susceptibility to organochlorine (DDT 2%), carbamate (propoxur 0.01%), organophosphate (malathion 2%, fenitrothion 1%, and pirimiphos methyl 1%), and pyrethroid (deltamethrin 0.06%, lambdacyhalothrin 0.06%, and permethrin 0.2%) insecticides. Susceptibility to the insecticides tested was evaluated in the field population of L. longipalpis and compared with a laboratory reference strain. The (LT95) to propoxur and malathion insecticides for the field population was lower than the LT95 for the laboratory reference strain, demonstrating high susceptibility to these compounds. A low level of resistance at LT50 (< 3-fold) was found for fenitrothion, pirimiphos methyl, and permethrin insecticides, but no resistance was detected at LT95. No significant resistance at the LT50 and LT95 was detected for the pyrethroids deltamethrin and lambdacyhalothrin. The susceptibility levels of L. longipalpis to the insecticides tested are discussed in view of a future control program against endophilic vectors of leishmaniases based on the use of pesticides.

Animals↗
PubMed1996

Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system.

A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family. Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin. Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity. Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions. Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.

Arginine↗
PubMed1996

Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis.

The sigE and sigK genes, encoding the sporulation-specific sigma factors sigma 35 and sigma 28 of Bacillus thuringiensis, were each disrupted by inserting a gene conferring resistance to kanamycin into their coding sequences. The B. thuringiensis SigE- and sigK- mutant strains were blocked at different sporulation stages and were unable to sporulate. The SigE-strain was blocked at stage II of sporulation, whereas the SigK- strain was blocked at stage IV. The expression of a cryIAa'-'lacZ transcriptional fusion was analysed in these genetic backgrounds and it was found that both sigma factors are involved in the in vivo transcription of this gene. However, the SigK- strain harbouring the cryIAa gene produced amounts of toxin similar to those produced by the B. thuringiensis Spo+ strain. The toxins accumulated in the mother cell compartment to form a crystal inclusion which remained encapsulated within the cell wall. Thus, transcription from the sigma E-dependent promoter alone (Bt I promoter) is sufficient to support high levels of toxin production in B. thuringiensis.

Bacillus thuringiensis↗
PubMed1996

Interactions of Bacillus thuringiensis crystal proteins with the midgut epithelial cells of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Binding of different Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles. Our results show that ICPs interact with the microvilli of epithelial cells of S. frugiperda in two different ways. The first is typical of highly toxic proteins (like CryIC and CryID); this interaction is saturable and specific. In contrast, some nontoxic proteins (like CryIAb) interact nonspecifically with the microvilli, since the binding of this toxin is not affected by the presence of high concentrations of homologous competitor. The CryIC toxin binds to two brush border proteins of 40 and 44 kDa and the CryIAb toxin binds to a single protein of 150 kDa. Immunological detection of ingested B. thuringiensis ICPs on gut sections of S. frugiperda larvae revealed that CryIC and CryID toxins bound along the epithelial brush border microvilli membrane. Binding of the nontoxic protein CryIAb was also observed in the epithelial brush border membrane of fed larvae, but it was extremely weak, implying that this type of interaction occurs also in vivo although it is not related to toxicity.

Animals↗
PubMed1996

Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis.

The Bacillus thuringiensis (Bt) cryIIIA gene is regulated by a different mechanism from that of most of the other cry genes. Its expression begins during late-exponential growth and not during sporulation as for the other classes of cry genes. Moreover, in Bacillus subtilis, cryIIIA expression is independent of the major sporulation-specific sigma factors and is increased in a spoOA genetic background. We used lacZ fusions and primer-extension analysis to follow the time-course of cryIIIA transcription in Bt wild-type and in various Spo- genetic backgrounds (spoOA, sigE and sigK). cryIIIA was activated from the end of vegetative growth to stage II of sporulation (t3) in the wild-type strain. Thereafter, transcription from the same promoter continued, at a decreasing rate, until the end of stage III. In the spoOA mutant strain, the same promoter was activated for at least 15 h during the stationary phase. cryIIIA activation in the sigK genetic background was similar to that in the wild-type but was extended in a sigma E mutant strain. Thus cryIIIA expression in Bt is not directly dependent on the major sporulation-specific sigma factors. Furthermore, an event linked with the thE-dependent period of sporulation ends cryIIIA activation, although transcription of this gene does not switch off before the end of stage III.

Bacillus thuringiensis↗
PubMed1995

Determination of anions in human and animal tear fluid and blood serum by ion chromatography.

An important factor contributing to the development of ion chromatography (IC) has been the need for repetitive analyses of samples with high ionic contents and samples available in microvolumes. IC was selected for the determination of Cl, NO3, SO(2-)4 and PO(3-)4 anions in tear fluid and serum from ten human volunteers of both sexes, seven young-adult black vultures (Coragyps atratus) and three young-adult chickens (Gallus gallus domesticus). The samples were analysed on a Dionex Model 2000i/SP ion-exchange chromatograph equipped with an anion guard column (Dionex IonPac AG4A), anion separator column (Dionex IonPac AS4A), suppressor column (Dionex AMMS-II) and a conductivity detector. The flow-rte of the mobile phase, 1.7 mM NaHCO3 - 1.8 mM Na2CO3 was set at 2.0 ml/min. The R.S.D. was calculated to be less than 1.5% for all anions. In the human, black vulture and chicken serum samples, the NO3, PO(3-)4 and SO(2-)4 anion contents were higher than in tears; for Cl the reverse was found. No correlation was found amongst the anion concentrations present in the tear fluid and blood serum in all samples (p > 0.05). With no sample treatment, column maintenance was required.

Adolescent↗
PubMed1995

A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferation-related induction in transgenic mice.

Keratins, the constituents of epithelial intermediate filaments, are precisely regulated in a tissue- and development-specific manner, although little is known about the molecular mechanisms underlying this regulation. The expression pattern of keratin 6 is particularly complex, since besides being constitutively expressed in hair follicles and in suprabasal cells of a variety of internal stratified epithelia, it is induced in epidermis in both natural and artificially caused hyperproliferative situations. Therefore, the regulatory sequences controlling keratin 6 gene activity are particularly suitable for target gene expression in a tissue-specific manner. More interestingly, they can be skin-induced in transgenic animals or in gene therapy protocols, particularly those addressing epidermal hyperproliferative disorders. To delimit the regions containing these regulatory elements, different parts of the bovine keratin 6 gene linked to a beta-galactosidase reporter gene have been assayed in transgenic mice. A 9-kbp fragment from the 5' upstream region was able to provide both suprabasal tissue-specific and inducible reporter expression.

Animals↗
PubMed1995

Delta-endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers.

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, not very K+ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2-40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis delta-endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV-containing functional receptors.

Animals↗
PubMed1995

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication.

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.

Bacillus Phages↗
PubMed1995

Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper.

Bacillus thuringiensis↗
PubMed1995

Tissue-specific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice.

Keratin 8 is a type II intermediate filament protein found in simple epithelia. We have introduced a 12 kb DNA fragment of the human K8 locus into the germ line of mice. The transgene, containing 1.1 kb of 5' flanking sequences, 7.7 kb corresponding to the body of the gene and 3.2 kb of 3' flanking sequences, was expressed in all six lines obtained. Immunolocalization and RNA analysis of adult tissues showed that the tissue-specific expression pattern of the transgene was almost indistinguishable from that of the endogenous gene. This pattern was found in organs containing single epithelial cell types, such as trachea, lung, stomach, intestine, liver, kidney, thymus and glands. The highest expressing line, however, also produced human K8 in tissues such as stratified epithelia, where it formed part of the pre-existing keratin cytoskeleton of basal cells. Steady state levels of human K8 RNA were proportional to the copy number of the transgene, but transgene expression was less efficient, per gene copy, than that of the endogenous gene. When in the 12 kb DNA fragment the exons and introns of the gene were replaced by the Escherichia coli lacZ gene, the resulting construct showed no expression in transgenic mice. This suggests that 5' and 3' flanking sequences, in the absence of intragenic sequences, are not sufficient for K8 expression and that important control elements are located in the body of the K8 gene.

Animals↗
PubMed1995

[Gastric cavernous hemangioma as a cause of upper digestive hemorrhage].

Upper digestive bleeding caused by an isolated gastric hemangioma is a rather rare occurrence, even in cases of Osler-Weber-Rendu syndrome. The case reported here, which brings to 40 the number reported in the world literature, is that of a 68-year-old patient who presented with upper digestive bleeding. Endoscopy led us to suspect a hemangioma due to the abnormally large lesion, a suspicion that was confirmed preoperatively through arteriography, which detected a hypervascular mass of 6 x 4 cm dependent on the gastroduodenal artery. This finding was in turn confirmed by the surgical intervention. Diagnostic difficulties based on barium studies, endoscopy and surgical exploration have meant that preoperative diagnosis has been rare in the cases published to date. We consider that a more liberal use of arteriography could reverse this tendency, as indeed our case has shown. Furthermore, we feel that arteriography is indicated in cases of upper digestive bleeding of obscure origin, which might be due to small angiomas that may be overlooked in other diagnostic tests.

Aged↗
PubMed1994

A genetic approach to the identification of functional amino acids in protein p6 of Bacillus subtilis phage phi 29.

Protein p6 of the Bacillus subtilis phage phi 29 is essential for in vivo viral DNA replication. This protein activates the initiation of phi 29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su) cells to support growth of a phi 29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.

Bacillus Phages↗
PubMed1994

In vivo functional relationships among terminal proteins of Bacillus subtilis phi 29-related phages.

Gene 3 of the Bacillus subtilis phage phi 29 encodes the terminal protein (TP), which acts as a primer in the initiation of viral DNA replication. We have developed an in vivo functional assay for the phi 29 TP based on the ability of TP-producing B. subtilis non-suppressor (su-) cells to support DNA replication of a phi 29 sus3 mutant phage. This trans-complementation assay has been used to study in vivo functional relationships between the TP of phi 29 and related phages. Our results demonstrate that phi 29 TP functionally substitutes the TP of phage PZA, whereas replication of phage Nf DNA cannot take place in vivo using the phi 29 TP as a primer.

Bacillus Phages↗
PubMed1994

Phage phi 29 protein p6: a viral histone-like protein.

Phage phi 29 protein p6 is one of the most abundant viral proteins in phi 29-infected B subtilis cells, constituting about 4% of the total cellular proteins (about 3 x 10(6) copies/cell) at late infection. Electron microscopic studies showed that, in vitro, protein p6 forms heterogeneously-sized complexes all along phi 29 DNA, suggesting that protein p6 may have a role in genome packaging and organization. The low stability of the protein p6-phi 29 DNA complexes observed in vitro could reflect the dynamic nature of these complexes, to allow replication, transcription, and encapsidation of the genome. The protein p6-DNA complex consists of a DNA right-handed superhelix wrapped around a multimeric protein core. The DNA in this complex is strongly distorted and compacted. Protein p6 recognition signals have been mapped near the ends of the linear phi 29 DNA and act as nucleation sites for complex formation. Protein p6 does not recognize a specific sequence, but sequences with specific bendable properties that would favor the formation of the complex. Protein p6 represses transcription from the phi 29 C2 early promoter, and activates initiation of phi 29 DNA replication that occurs from both DNA ends. The formation of nucleoprotein complexes at the origins of replication, as well as the specific positioning of protein p6 with respect to the DNA ends are required for the activation of replication. This suggests that the proteins involved in the initiation step of phi 29 DNA replication, either directly interact with protein p6, or recognize a conformational change at a specific location in the DNA. The mechanism of activation could be the local and transient unpairing of DNA at specific sites, facilitated by the strong distortion of DNA conformation in the nucleoprotein complex.

Bacillus Phages↗