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Comparison of the activities of two allelic variants of the human wildtype p53 protein
No full textThe human wildtype p53 tumor suppressor gene contains a polymorphism at amino acid residue 72 which results in either an arginine (p53 Arg-72) or proline (p53 Pro-72) at this codon. In the present study I have examined this polymorphism at the molecular level to determine whether differences exist in the biochemical functions of these two p53 variants. No differences were observed in their sequence-specific DNA binding abilities, nor in their ability to be targeted by HPV-18 E6 oncoprotein for degradation by ubiquitination in vitro. However, differences were observed in the ability of these two variants to function as transcriptional activators: p53 Pro-72 was more transcriptionally active than p53 Arg-72. I propose that the polymorphism at codon 72 may affect the structure of the N-terminal transactivation domain of the p53 protein, which would then have an effect on the ability of these variants to interact with transcription factors in order to initiate transcription of target genes and function as a tumor suppressor
Induction of apoptosis or cell cycle arrest by two human wildtype variants of the p53 protein
No full textThe human wildtype p53 tumor suppressor gene is found in two different forms, p53 Arginine and p53 Proline. This difference results in a substitution of a proline for an arginine at codon 72 producing the polymorphism. Knowing that apoptosis and cell cycle arrest are the two main functions of p53, the objective of this project was to determine the difference in the capacity of these allelic variants of p53 to induce apoptosis and/or cell cycle arrest in two different experimental model systems. The first experimental system was composed of non-transformed 10(1) cells and the second one was transformed Saos-2 cells. In the first experimental system, the two wildtype forms of p53 induced cell cycle arrest at the same level and did not induce apoptosis. On the other hand, in transformed cells, both p53Arg and p53Pro induced apoptosis at similar levels. No cell cycle arrest activity has been detected in Saos-2 cells. In conclusion, this study suggests that the induction of cell cycle arrest or apoptosis depends more on the cell type than on the type of the p53 protein. Also, the intensity of cell cycle arrest or apoptosis is independent of which allelic variant of p53 is present under the experimental conditions used in this study
Molecular studies using amastigote-specific genes in Leishmania
No full textLeishmania is a dimorphic parasitic protozoan which exists as a flagellated promastigote in the sandfly vector and as an intracellular amastigote in the phagolysosomal compartment of mammalian host macrophages. It is the amastigote form that is responsible for the pathology in susceptible vertebrate hosts. Leishmania donovani is responsible for visceral leishmaniasis, the most severe form of the leishmanial diseases. We have investigated the antibody response against an amastigote-specific protein, A2, which is developmentally expressed in L. donovani during promastigote-to-amastigote cytodifferentiation. A2 is conserved in L. donovani and L. mexicana species but not in other Leishmania species tested. We have shown that this characteristic contributes to its potential as a useful specific diagnostic antigen for visceral leishmaniasis. Developmental expression of A2 involves A2 mRNA untranslated regions (UTRs) and we have demonstrated that A2 UTRs can regulate expression of exogenous suicide genes throughout the Leishmania life cycle. We have shown that the A2 gene regulatory system has potential for the generation of developmentally attenuated L. donovani strains. Finally, we have performed a preliminary characterization of a gene, A2rel, that is tandemly associated with A2 genes in the genome. Contrary to A2 genes, the A2rel gene is well conserved in the Leishmania species. Although A2rel does not share sequence similarity with any known leishmanial genes characterized to date, it does appear to share characteristics with membrane-bound glycoproteins
Studies on molecular mechanisms of transformation by human papillomavirus : the role of E6 and E5 oncogenes
No full textThe ability of the HPV-18 E6 gene to impair p53-mediated transcriptional activity induced by DNA damaging agents was investigated. It is demonstrated that E6 can abolish DNA damage induced p53-mediated transcription and that a region from amino acid residue 113 to 117 of HPV-18 E6 protein was necessary for E6 to direct the degradation of p53. The biological importance of the E6/p53 interaction was then directly examined in HPV-16 containing cervical carcinoma derived cells by introducing the monomeric p53 mutant which is resistant to E6 mediated degradation. The two major observations made from this study were: (i) loss of p53 activity plays an important role in maintaining the malignant phenotype of these cells with respect to cell proliferation; (ii) the monomeric p53 mutant without its C-terminal regulatory region was biologically functional with respect to impairing cell proliferation in HPV-16 containing cervical carcinoma derived cells. Finally, it was revealed that the cellular MAP kinase signal transduction pathway was more active in cells expressing the HPV-16 E5 gene than in control cells or cells expressing E6 and E7. These observations help to define the mechanisms by which HPV oncogenes contribute to the development and maintenance of the neoplastic phenotype
Involvement of tyrosine phosphorylation during Leishmania donovani differentiation
No full textDimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms
Regulation of macrophage function during intracellular infection with Leishmania donovani
No full textInvestigation of the molecular alterations of macrophage function during intracellular infection by Leishmania donovani revealed both adverse and positive influences of this protozoan on host cell function. Chapter I delineates a negative effect which this parasite has on signal transduction pathways in its host cell. In macrophages put in contact, or infected with L. donovani, c-fos gene expression mediated through protein kinase A was unaffected under conditions where there was an impairment of protein kinase C-mediated c-fos gene expression. Selective impairment of protein kinase C-, or, calmodulin-dependent protein kinase-mediated signal transduction in the macrophage was found to influence the establishment of infection. Chapters two and three describe a positive enhancement of macrophage function by L. donovani. Intramacrophage infection with L. donovani was shown to enhance host cell viability in the absence of growth factor. This was attributable to the elaboration of a soluble factor(s) by infected macrophages into the cell culture supernatant, which enhanced macrophage viability in a manner independent of cell replication. Further characterization of the mechanism of this enhancement revealed that L. donovani infection, and lipophosphoglycan, inhibited macrophage death by apoptosis. Cell supernatants derived from L. donovani infected cells were also capable of inhibiting macrophage apoptosis. To identify the active factor in infected cell supernatants, the cytokine gene expression profile of L. donovani infected macrophages was delineated and possible candidate cytokines were further investigated. Levels of TNF- capable of causing an abrogation of apoptosis were found to be produced by infected macrophages. However, antibody neutralization of TNF in infected cell cultures could not reverse the inhibition of apoptosis by L. donovani, implicating the involvement of multiple factors in the abrogation of apoptosis by L. donovani
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Trypanosoma cruzi CYP51 inhibitor derived from a Mycobacterium tuberculosis screen hit.
Get PDFBACKGROUND:The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14alpha-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols. METHODOLOGY/PRINCIPAL FINDING:In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51(Mt)), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51(Mt). Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51(Tc), demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti-T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site. CONCLUSIONS/SIGNIFICANCE:CYP51(Mt)-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51(Tc)
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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