The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate

Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.

Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.

Topological analysis of the Escherichia coli WcaJ protein reveals a new conserved configuration for the polyisoprenyl-phosphate hexose-1-phosphate transferase family

WcaJ is an Escherichia coli membrane enzyme catalysing the biosynthesis of undecaprenyl-diphosphate-glucose, the first step in the assembly of colanic acid exopolysaccharide. WcaJ belongs to a large family of polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) sharing a similar predicted topology consisting of an N-terminal domain containing four transmembrane helices (TMHs), a large central periplasmic loop, and a C-terminal domain containing the fifth TMH (TMH-V) and a cytosolic tail. However, the topology of PHPTs has not been experimentally validated. Here, we investigated the topology of WcaJ using a combination of LacZ/PhoA reporter fusions and sulfhydryl
labelling by PEGylation of novel cysteine residues introduced into a cysteine-less WcaJ. The results showed that the large central loop and the C-terminal tail both reside in the cytoplasm and are separated by TMH-V, which does not fully span the membrane, likely forming a "hairpin" structure. Modelling of TMH-V revealed that a highly conserved proline might contribute to a helix-break-helix structure in all PHPT members. Bioinformatic analyses show that all of these features are conserved in PHPT homologues from
Gram-negative and Gram-positive bacteria. Our data demonstrate a novel topological configuration for PHPTs, which is proposed as a signature for all members of this enzyme family

“If I Were Nick”: Men’s Responses to an Interactive Video Drama Series to Support Smoking Cessation

Background: Men continue to smoke in greater numbers than women; however, few interventions have been developed and tested to support men’s cessation. Men also tend to rely on quitting strategies associated with stereotypical manliness, such as willpower, stoicism and independence, but may lack the self‐efficacy skills required to sustain a quit. In this article we describe the development of and reception to an interactive video drama (IVD) series, composed of 7 brief scenarios, to support and strengthen men’s smoking cessation efforts. The value of IVD in health promotion is predicated on the evidence that viewers engage with the material when they are presented characters with whom they can personally identify. The video dramatizes the challenges unfolding in the life of the main character, Nick, on the first day of his quit and models the skills necessary to embark upon a sustainable quit. 
Objective: The objective was to describe men’s responses to the If I were Nick IVD series as part of a pilot study of QuitNow MenTM, an innovative smoking cessation website designed for men. Specific objectives were to explore the resonance of the main character of the IVD series with end‐users, and men’s perceptions of the effectiveness of the IVD series for supporting their quit self‐management. 
Methods: Seven brief IVD scenarios were developed, filmed with a professional actor and uploaded to a new online smoking cessation website, QuitNow MenTM.  A sample of 117 men who smoked were recruited into the study and provided baseline data prior to access to the QuitNow MenTM website for a 6 month period. During this time, 47 men chose to view the IVDs. Their responses to questions about the IVDs were collected in 3‐month and 6‐month online follow‐up surveys and analyzed using descriptive statistics. 
Findings: The majority of participants indicated they related to the main character, Nick. Participants who “strongly agreed” they could relate to Nick perceived significantly higher levels of support from the IVDs than the “neutral” and “disagree” groups (P <.001, d =2.0, P <.001 d =3.1). The “agree” and “neutral” groups were significantly higher on rated support from the videos than the “disagree” (P <.001 d =2.2, P =.01 d = 1.5). Participants’ perception of the main character was independent of participant age, education attainment or previous quit attempts. 
Conclusions: The findings suggest that IVD interventions may be an important addition to men’s smoking cessation programs. Given that the use of IVD scenarios in health promotion is in its infancy, the positive outcomes from this pilot study signal the potential for IVD and warrant ongoing evaluation in smoking cessation and, more generally, men’s health promotion.  

Selective induction of apoptosis by the pyrrolo-1,5-benzoxazepine 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) in Leukemia cells occurs via the c-Jun NH2-terminal kinase-dependent phosphorylation and inactivation of Bcl-2 and Bcl-XL

Overexpression of the Bcl-2 proto-oncogene in tumor cells confers resistance against chemotherapeutic drugs. In this study, we describe how the novel pyrrolo-1,5-benzoxazepine compound 7-[[dimethylcarbamoyl]oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine (PBOX-6) selectively induces apoptosis in Bcl-2-overexpressing cancer cells, whereas it shows no cytotoxic effect on normal peripheral blood mononuclear cells. PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis in chronic myelogenous leukemia (CML) K562 cells by the time- and dose-dependent phosphorylation and inactivation of antiapoptotic Bcl-2 family members Bcl-2 and Bcl-XL. PBOX-6 also induces Bcl-2 phosphorylation and apoptosis in wild-type T leukemia CEM cells and cells overexpressing Bcl-2. This is in contrast to chemotherapeutic agents such as etoposide, actinomycin D, and ultraviolet irradiation, whereby overexpression of Bcl-2 confers resistance against apoptosis. In addition, PBOX-6 induces Bcl-2 phosphorylation and apoptosis in wild-type Jurkat acute lymphoblastic leukemia cells and cells overexpressing Bcl-2. However, Jurkat cells containing a Bcl-2 triple mutant, whereby the principal Bcl-2 phosphorylation sites are mutated to alanine, demonstrate resistance against Bcl-2 phosphorylation and apoptosis. PBOX-6 also induces the early and transient activation of c-Jun NH2-terminal kinase (JNK) in CEM cells. Inhibition of JNK activity prevents Bcl-2 phosphorylation and apoptosis, implicating JNK in the upstream signaling pathway leading to Bcl-2 phosphorylation. Collectively, these findings identify Bcl-2 phosphorylation and inactivation as a critical step in the apoptotic pathway induced by PBOX-6 and highlight its potential as an effective antileukemic agent.

Novel functional interaction between the plasma membrane Ca2+ pump 4b and the proapoptotic tumor suppressor Ras-associated factor 1 (RASSF1)

Plasma membrane calmodulin-dependent calcium ATPases (PMCAs) are enzymatic systems implicated in the extrusion of calcium from the cell. We and others have previously identified molecular interactions between the cytoplasmic COOH-terminal end of PMCA and PDZ domain-containing proteins. These interactions suggested a new role for PMCA as a modulator of signal transduction pathways. The existence of other intracellular regions in the PMCA molecule prompted us to investigate the possible participation of other domains in interactions with different partner proteins. A two-hybrid screen of a human fetal heart cDNA library, using the region 652-840 of human PMCA4b (located in the catalytic, second intracellular loop) as bait, revealed a novel interaction between PMCA4b and the tumor suppressor RASSF1, a Ras effector protein involved in H-Ras-mediated apoptosis. Immunofluorescence co-localization, immunoprecipitation, and glutathione S-transferase pull-down experiments performed in mammalian cells provided further confirmation of the physical interaction between the two proteins. The interaction domain has been narrowed down to region 74-123 of RASSF1C (144-193 in RASSF1A) and 652-748 of human PMCA4b. The functionality of this interaction was demonstrated by the inhibition of the epidermal growth factor-dependent activation of the Erk pathway when PMCA4b and RASSF1 were co-expressed. This inhibition was abolished by blocking PMCA/RASSSF1 association with an excess of a green fluorescent protein fusion protein containing the region 50-123 of RASSF1C. This work describes a novel protein-protein interaction involving a domain of PMCA other than the COOH terminus. It suggests a function for PMCA4b as an organizer of macromolecular protein complexes, where PMCA4b could recruit diverse proteins through interaction with different domains. Furthermore, the functional association with RASSF1 indicates a role for PMCA4b in the modulation of Ras-mediated signaling.

A peptide inhibitor of C-jun N-terminal kinase modulates hepatic damage and the inflammatory response after hemorrhagic shock and resuscitation

Hemorrhage and resuscitation (H/R) leads to phosphorylation of mitogen-activated stress kinases, an event that is associated with organ damage. Recently, a specific, cell-penetrating, protease-resistant inhibitory peptide of the mitogen-activated protein kinase c-JUN N-terminal kinase (JNK) was developed (D-JNKI-1). Here, using this peptide, we tested if inhibition of JNK protects against organ damage after H/R. Male Sprague-Dawley rats were treated with D-JNKI-1 (11 mg/kg, i.p.) or vehicle. Thirty minutes later, rats were hemorrhaged for 1 h to a MAP of 30 to 35 mmHg and then resuscitated with 60% of the shed blood and twice the shed blood volume as Ringer lactate. Tissues were harvested 2 h later. ANOVA with Tukey post hoc analysis or Kruskal-Wallis ANOVA on ranks, P < 0.05, was considered significant. c-JUN N-terminal kinase inhibition decreased serum alanine aminotransferase activity as a marker of liver injury by 70%, serum creatine kinase activity by 67%, and serum lactate dehydrogenase activity by 60% as compared with vehicle treatment. The histological tissue damage observed was blunted after D-JNKI-1 pretreatment both for necrotic and apoptotic cell death. Hepatic leukocyte infiltration and serum IL-6 levels were largely diminished after D-JNKI-1 pretreatment. The extent of oxidative stress as evaluated by immunohistochemical detection of 4-hydroxynonenal was largely abrogated after JNK inhibition. After JNK inhibition, activation of cJUN after H/R was also reduced. Hemorrhage and resuscitation induces a systemic inflammatory response and leads to end-organ damage. These changes are mediated, at least in part, by JNK. Therefore, JNK inhibition deserves further evaluation as a potential treatment option in patients after resuscitated blood loss.

Diferencias en costo efectividad del cuidado paliativo domiciliario frente a la atención convencional hospitalaria para el manejo de enfermedades crónicas en fase terminal

En Colombia la dinámica poblacional ha experimentado cambios a través de los años; lo cual modifica las características epidemiológicas y los perfiles de morbimortalidad de la población, aumentando la incidencia y prevalencia de enfermedades crónicas de difícil pronóstico dentro de la población; siendo estos grupos etarios hacía quienes deben destinarse nuevas modalidades de atención y soporte paliativo. Se realizó una revisión sistemática de diversos artículos, clasificados con niveles de evidencia I y II; cuyas variables de análisis fueron: El lugar de fallecimiento del paciente, alivio de la sintomatología, nivel de satisfacción del paciente y sus cuidadores con los servicios recibidos, número de ingresos hospitalarios independientemente de la causa y días promedio de estancia hospitalaria. El 88% de los pacientes que fueron vinculados a la atención domiciliaria falleció en su hogar; el 68% refirió haber experimentado disminución en la intensidad de al menos 2 síntomas como resultado de la atención en casa. El nivel de satisfacción para el cuidado en casa puntuó 19 puntos porcentuales por encima de la calificación que obtuvo el manejo hospitalario. El 23% de los pacientes tratados en su hogar, requirió al menos una hospitalización durante el manejo; mostrando una disminución en el tiempo de estancia hospitalaria de 3.4 días promedio. El manejo paliativo domiciliario de pacientes crónicos en fase terminal, no sólo es una alternativa en la reducción de costos para el sistema de salud, sino que es un factor protector de tipo emocional que refleja también una importante disminución de los riesgos de la hospitalización.

Un projecte d'orientació per a la fase terminal dels ensenyaments bàsics. 'Un proyecto de orientación para la fase terminal de las enseñanzas básicas'.

Ofrecer unas aportaciones prácticas a todos los interesados en hacer orientación. La orientación. La adolescencia. Para el trabajo práctico se cuenta con una muestra de 200 alumnos que el curso 1977-78 cursaron octavo de EGB en colegios nacionales de la provincia de Tarragona. Hacen una exposición teórica sobre la orientación. Hacen un estudio teórico de la adolescencia. Exponen un método específico para estudiar las baremaciones de los tests creados por uno de los autores de la tesina. Analizan las baremaciones de las siguientes pruebas: TIL, PMA, DAT, ACH-73, CEP, Atención (Salamanca). Hacen un estudio del test de Preferencias Profesionales RMI, del test de Intereses Profesionales Gonzalo-Luján y del test de Adaptación para Adolescentes Bell. Exponen el proyecto de orientación. Bibliografía. Batería de pruebas que pasa el Servei Provincial d'Orientació Escolar i Vocacional de Tarragona. Método específico ad hoc para estudiar las baremaciones de los tests. El tutor no es la persona idónea para dedicarse a la orientación. Se propone la creación de la especialidad de Orientador en la Facultad de Ciencias de la Educación. El modelo propuesto permite crear y hacer funcionar un Departamento de Orientación con un mínimo de personal y coste y con buena rentabilidad.

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG]2 with a topoisomerase poison in the presence of Co2+Ions†,‡

The structure of the duplex d[CG(5-BrU)ACG]2 bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 Å resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co2+ ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40°, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co2+ ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA (‘hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.

Local orientational disorder in peptide fibrils probed by a combination of residue-specific 13C-18O labelling, polarised infrared spectroscopy and molecular combing

A novel combination of site-specific isotope labelling, polarised infrared spectroscopy and molecular combing reveal local orientational ordering in the fibril-forming peptide YTIAALLSPYSGGRADS. Use of 13C-18O labelled alanine residues demonstrates that the Nterminal end of the peptide is incorporated into the cross-beta structure, while the C-terminal end shows orientational disorder

Terminal: a miracle play with popular music from the end of the world

Terminal: A Miracle Play with Popular Music from the End of the World is a film and live performance project exploring the politics of post-apocalyptic fiction. A theatrical staging of a morality play for end times and future folk music, it recasts eschatology, as a foundational myth for a future society. Post-apocalyptic writing and cinema are grounded in an ethos of survivalism. Invoking Rousseau’s state of nature, or time before government, these fictions propose violent scenarios in which nuclear holocaust, environmental catastrophe and other disasters generate an individualistic politics of pure pragmatism, negating the possibility of democratic deliberation. Terminal narrates this familiar scenario, but at the same time questions its validity. The film, shot on black and white VHS at Kurt Schwitters’ Merzbarn in Cumbria, dramatises a series of conversations between future-historical archetypes about the needs and pressures of the situation in which they find themselves at the end of the world. The performers then gather to play worshipful songs about acid rain, radiation sickness and eating the dog, using a mix of conventional, obscure and makeshift instruments In the tradition of books such as Russell Hoban’s Riddley Walker and Arthur M. Miller Jr.’s A Canticle for Liebowitz, Terminal imagines artistic expression and new folk traditions for a world to come after the apocalypse. If, as Slavoj Žižek would have it, it is easier to imagine the end of the world than to think of the end of capitalism, the project juxtaposes these two endpoints to test out how alternative scenarios might emerge from the collaborative practice of making theatre and music against a setting of social collapse.

The differential roles of D-Pax2 variants in regulating Drosophila eye and bristle development

The ability to appropriately interact with the environment is crucial to an organism’s survival. The establishment of functional sensory systems, such as the bristles and eyes in Drosophila, is a critical event during the development of the organism. The transcription factor D Pax2 is involved in the differentiation of the shaft and glial cells in the developing bristle (Kavaler et al., Dev, 126:2261-2272, 1999) and of the cone and primary pigment cells in the developing eye (Fu and Noll, Genes Dev, 11:389-405, 1997). How D-Pax2 contributes to distinct differentiative pathways in different cell types is not known. Recent work by Anna Czechowski and Katherine Harmon (personal communication) identified a mutation in the D-Pax2 gene that introduced a stop codon at the end of exon 9, effectively truncating the protein. This mutation affects bristle, but not eye, development. We thus suspected regions after exon 9 are required for D-Pax2 function only in the bristles and may also be associated with alternative splicing of the D Pax2 transcript. We plan to assess the role of the carboxy terminal region of the protein by establishing transgenic lines bearing rescue constructs of D-Pax2 with either the complete coding sequence or with deletions of specific exons. To date, we have generated the first rescue construct bearing the complete coding region of the gene driven by a 3 KB upstream regulatory region of D-Pax2 and are currently generating transgenic fly lines with this construct.

Efeito da suplementação de vitamina D no desenvolvimento de lesões pré-neoplásicas quimicamente induzidas em ratos Wistar macho

Pós-graduação em Biologia Geral e Aplicada - IBB

Variabilidade do padrão de esterases e atividade da glutationa S-transferase em linhagens geográficas de Drosophila melanogaster e D. simulans resistentes e suscetíveis ao inseticida DDT

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)