Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis.

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65(-/-) mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

Increased plasma levels of N-terminal brain natriuretic peptide (NT-proBNP) in type 2 diabetic patients with vascular complications.

AIMS: The plasma levels of either brain natriuretic peptide (BNP) or the N-terminal fragment of the prohormone (NT-proBNP) have recently gained extreme importance as markers of myocardial dysfunction. Patients with type 2 diabetes are at high risk of developing cardiovascular complications. This study was aimed to assess whether plasma NT-proBNP levels are at similar levels in type 2 diabetics with or without overt cardiovascular diseases. METHODS: We assayed plasma NT-proBNP in 54 type 2 diabetics, 27 of whom had no overt macro- and/or microvascular complications, while the remaining ones had either or both. The same assay was carried out in 38 healthy control subjects age and sex matched as a group with the diabetics. RESULTS: Plasma NT-proBNP was higher in diabetics (median 121 pg/ml, interquartile range 50-240 pg/ml, ) than in those without complications (37 pg/ml, 21-54 pg/ml, P<0.01). Compared with the controls (55 pg/ml, 40-79 pg/ml), only diabetics with vascular complications had significantly increased plasma NT-proBNP levels (P<0.001). In the diabetics, coronary heart disease and nephropathy (defined according to urinary excretion of albumin) were each independently associated with elevated values of plasma NT-proBNP. CONCLUSIONS: In type 2 diabetes mellitus, patients with macro- and/or micro-vascular complications exhibit an elevation of plasma NT-proBNP levels compared to corresponding patients with no evidence of vascular disease. The excessive secretion of this peptide is independently associated with coronary artery disease and overt nephropathy. The measurement of circulating NT-proBNP concentration may therefore be useful to screen for the presence of macro- and/or microvascular disease.

Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

Optimización de la distribución de LNG en una red paneuropea de transporte pesado de mercancías de larga distancia por carretera por medio de programación lineal

Actualmente, el transporte por carretera de mercancías de larga distancia está dominado por vehículos propulsados por diésel. Este combustible fósil derivado del petróleo puede ser parcialmente sustituido por otro combustible alternativo: el gas natural licuado (LNG). Su uso proporcionará una disminución de la dependencia de los estados productores de petróleo, es comparable en energía específica y densidad energética al diésel, es seguro, disminuye las emisiones contaminantes, en nivel de ruido y es actualmente más económico. Recopilar datos reales sobre el transporte por carretera en la EU27 (energía utilizada por este tipo de transporte, localización de las terminales de LNG operativas, determinar las reservas disponibles, calcular la población que va a cubrir y calcular la energía necesaria disponible para dar cobertura a esa población según la cantidad de energía per cápita utilizada para este tipo de transporte). Determinar las interconexiones a cubrir por la red paneuropea de transporte propuesta y decidir las localizaciones de las EESS a ser construidas para cubrir las recomendaciones de la EC de manera que ningún tramo supere los 400 km dentro de esos itinerarios. Calcular todas las distancias que separan cada terminal de LNG operatica de cada EESS (1300 distancias calculadas). Construir el modelo de programación lineal. Resolver, interpretar, validar y comprobar que es posible su utilización para la puesta en práctica del modelo propuesto, asumiendo la distribución total del LNG disponible y la minimización de los costes globales de distribución del mismo, desde el punto de vista económico. Se procederá buscando los datos en fuentes fiables que nos permitan realizar los cálculos necesarios para construir el modelo y resolviéndolo con el software adecuado a un modelo de estas dimensiones. Se ha conseguido una solución óptima, con la distribución total del LNG y minimizando los costes globales. Además, se ha obtenido movimiento de combustible en 76 de las 1300 variables, que han mostrado un déficit de terminales de almacenamiento de LNG para su distribución en el norte y centro de Europa, que se ve compensando con un flujo de transporte de este combustible desde la península ibérica principalmente y, en menor medida, desde Italia.

Implantació d'una plataforma informàtica en una entitat financera fusionada

Els darrers anys han estat molt convulsos en el món financer. De tots és coneguda la crisi, immobiliària en un primer moment, i financera més tard. Reestructuracions, fusions, absorcions, intervencions i nacionalitzacions han estat a l’ordre del dia en el món de les entitats financeres els últims 3 anys. El mapa financer ha passat de més de 50 entitats a poc menys de 10 en tan sols 3 anys. Els departaments de tecnologia de bancs i caixes no han estat aliens a aquests moviments. Tot i estar relativament al marge del negoci financer de l’entitat, cada fusió ha atorgat un paper protagonista a tota la plantilla informàtica. Ja que cada reestructuració ha suposat un gran esforç per integrar entitats molt diferents entre si en una de sola. Intentant que l’impacte a clients i a usuaris fos el menor possible. Caixa Manlleu és un exemple proper de la reestructuració del món financer, i ha protagonitzat, juntament amb Caixa Sabadell i Caixa Terrassa, la creació d’Unnim. Aquest cas real serà la base del treball. El projecte consisteix en la creació d’una plataforma informàtica per a la nova entitat resultant de la fusió. Una plataforma amb un doble objectiu: d’una banda, que reculli el millor de les tres plataformes d’origen i que compleixi tots els requestis que la nova entitat exigeix per tal que els usuaris puguin continuar fent les tasques diàries. I de l’altra, una plataforma que posi al dia i actualitzi tecnològicament l’entitat amb el mínim cost possible. Així doncs, cal analitzar per separat cadascuna de les 3 plataformes existents, inventariar les aplicacions comunes i les departamentals, així com les llicències de software vigents existents i no caducades. Decidir quines aplicacions es faran servir, si se’n poden aprofitar o si per contra en calen de noves. També cal analitzar quins equips informàtics (terminals) es necessitaran per tal de treure el màxim rendiment de la plataforma. Entre les conclusions obtingudes en el projecte, cal destacar els grans avantatges d’utilitzar aplicacions virtualitzades en lloc de les tradicionals aplicacions instal·lades físicament a cada terminal. Faciliten la feina i el manteniment, estalvien recursos i fan guanyar temps. A més a més s’aconsegueix una plataforma àgil, lleugera i ràpida.

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein.

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway.

OBJECTIVE: The pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) generates pancreatic beta-cells apoptosis mainly through activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. This study was designed to investigate whether the long-acting agonist of the hormone glucagon-like peptide 1 (GLP-1) receptor exendin-4 (ex-4), which mediates protective effects against cytokine-induced beta-cell apoptosis, could interfere with the JNK pathway. RESEARCH DESIGN AND METHODS: Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1 beta. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei. RESULTS: Ex-4 inhibited induction of the JNK pathway elicited by IL-1 beta. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1 beta. CONCLUSIONS: The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1 beta and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity.

The N-terminal domain of the circumsporozoite protein (CSP) has been largely neglected in the search for a malaria vaccine in spite of being a target of inhibitory antibodies and protective T cell responses in mice. Thus, in order to develop this region as a vaccine candidate to be eventually associated with other candidates and, in particular, with the very advanced C-terminal counterpart, synthetic constructs representing N- and C-terminal regions of Plasmodium falciparum and Plasmodium berghei CSP were administered as single or combined formulations in mice. We show that the antisera generated against the combinations inhibit sporozoite invasion of hepatocytes in vitro better than antisera against single peptides. Furthermore, two different P. falciparum CSP N-terminal constructs (PfCS22-110 and PfCS65-110) were recognized by serum samples from people living in malaria-endemic regions. Importantly, recognition of the short N-terminal peptide (PfCS65-110) by sera from children living in a malaria-endemic region was associated with protection from disease. Taken together, these results underline the potential of using such fragments as malaria vaccine candidates.

Visualització i control d'un pont de rentat de vehicles mitjançant un terminal HMI

Aquest treball pretén fer una anàlisi per millorar el control d'una màquina de rentar vehicles fent ús d'un terminal HMI junt a un autòmat. La realització de l'estudi d'usabilitat s'ha portat a terme seguint les etapes del disseny centrat en l'usuari, així com la identificació dels avantatges o inconvenients que pugui aportar aquest nou sistema.

Communication with terminal cancer patients in palliative care: are there differences between nurses and physicians?

The aim of this study was to find whether there were interprofessional differences in specific elements of communication with terminal cancer patients and decision-making processes that concern such patients. Given that interdisciplinary team work is one of the basic values in palliative care, if there are conflicting views between professions on such important issues it is most important to know about these and to understand them. A questionnaire utilized in an earlier survey of palliative care physicians and addressing their attitudes to and beliefs about specific elements of communication and decision making was sent to a sample of palliative care nurses working in the same regions, i.e. the French-speaking parts of Switzerland, Belgium and France. After a second mailing (reminder), 135 of the 163 questionnaires (83%) were returned. There was general agreement between nurses and physicians on questions dealing with perceptions of patients' knowledge of their diagnosis and stage of disease, patients' need for information, "do not resuscitate" orders and ethical principles in decision-making processes. Statistically significant, but small, differences between professional groups were only observed for a minority of the questions. Interprofessional differences in specific elements of communication with terminal cancer patients and decision-making processes affecting these patients were not so marked that they could be called "conflicting interprofessional views."

Nuclear localization of mouse fibroblast growth factor 2 requires N-terminal and C-terminal sequences.

In vertebrates, different isoforms of fibroblast growth factor 2 (FGF2) exist, which differ by their N-terminal extension. They show different localization and expression levels and exert distinct biological effects. Nevertheless, genetic inactivation of all FGF2 isoforms in the mouse results in only mild phenotypes. Here, we analyzed mouse FGF2, and show that, as in the human, mouse FGF2 contains CTG-initiated high molecular-weight (HMW) isoforms, which contain a nuclear localization signal, and which mediate localization of this isoform to the nucleus. Using green fluorescent protein-FGF2 fusions, we furthermore observed, that C-terminal deletions disable nuclear localization of the short low-molecular-weight (LMW) 18-kDa isoform. This loss of specific localization is accompanied by a loss in heparin binding. We therefore suggest that, first, localization of mouse FGF2 is comparable to that in other vertebrates and, second, FGF2 contains at least two sequences important for nuclear localization, a nuclear localization sequence at the N terminus which is only contained in the HMW isoform, and another sequence at the C terminus, which is only required for localization of the LMW 18-kDa isoform.

Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity.

Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]0.5) and high cooperativity toward this substrate. We have isolated mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa. Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphosphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.

Propuesta de muestreo para Neohydatothrips signifer (Thysanoptera: Thripidae) en el cultivo de maracuyá

El objetivo de este trabajo fue desarrollar una propuesta de muestreo de Neohydatothrips signifer en el cultivo de maracuyá. Se correlacionó la infestación promedio del trips en el cultivo con las variables meteorológicas de humedad relativa, temperatura y precipitación, con uso del coeficiente de correlación de Pearson. Así mismo, se determinó el índice de agregación de la población en el cultivo, con la prueba chi cuadrado (X²) para la distribución de Poisson, la prueba de la razón V²/µ, y la distribución de la binomial negativa. La única variable climática que se correlacionó significativamente con la densidad promedio del trips fue la temperatura. Se presentó mayor incidencia y densidad poblacional del insecto en los terminales vegetativos en comparación con los botones florales. El patrón de disposición espacial que se presentó en todos los estados de desarrollo fenológico del cultivo fue de tipo agregado. Con el índice de agregación y el tamaño promedio de la población, se calculó que se deben muestrear 22 terminales nuevos por hectárea, sin importar su posición en la planta. El muestreo del trips en el cultivo de maracuyá debe hacerse sobre los terminales vegetativos de la planta, sin importar la posición del terminal en ella.

A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss.

Hearing loss can be caused by a variety of insults, including acoustic trauma and exposure to ototoxins, that principally effect the viability of sensory hair cells via the MAP kinase (MAPK) cell death signaling pathway that incorporates c-Jun N-terminal kinase (JNK). We evaluated the otoprotective efficacy of D-JNKI-1, a cell permeable peptide that blocks the MAPK-JNK signal pathway. The experimental studies included organ cultures of neonatal mouse cochlea exposed to an ototoxic drug and cochleae of adult guinea pigs that were exposed to either an ototoxic drug or acoustic trauma. Results obtained from the organ of Corti explants demonstrated that the MAPK-JNK signal pathway is associated with injury and that blocking of this signal pathway prevented apoptosis in areas of aminoglycoside damage. Treatment of the neomycin-exposed organ of Corti explants with D-JNKI-1 completely prevented hair cell death initiated by this ototoxin. Results from in vivo studies showed that direct application of D-JNKI-1 into the scala tympani of the guinea pig cochlea prevented nearly all hair cell death and permanent hearing loss induced by neomycin ototoxicity. Local delivery of D-JNKI-1 also prevented acoustic trauma-induced permanent hearing loss in a dose-dependent manner. These results indicate that the MAPK-JNK signal pathway is involved in both ototoxicity and acoustic trauma-induced hair cell loss and permanent hearing loss. Blocking this signal pathway with D-JNKI-1 is of potential therapeutic value for long-term protection of both the morphological integrity and physiological function of the organ of Corti during times of oxidative stress.