Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl

Superposición de hepatitis B a hepatitis autoinmune. Diagnóstico y tratamiento, presentación de un caso clínico y revisión de casos clínicos en la literatura internacional

The hepatitis B virus represents one of the most frequent viral infections worldwide due to its easy transmission, in addition to this, its association with autoimmune pathologies has been found. This work will allow us to know the best treatment and most appropriate diagnostic method for its favorable clinical resolution. This systematic review was prepared in the database of Latin American and Caribbean Literature in Health Sciences, PubMed, and Google Scholar. The search was limited to studies carried out in humans, full texts, regardless of the language of writing or restriction of country of origin. A total of 133 articles were retrieved in the bibliographic search, of which 11 were excluded because they were duplicates, 71 articles were discarded because they contained brief information or only an ephemeral mention of the topic was made, 15 more were dispensed with in the Full review phase Text, 5 more for not being studies in humans and, finally, 10 opinions and perspectives, from which it was concluded that the treatment should be administered encompassing both pathologies to avoid clinical exacerbationEl virus de la hepatitis B representa una de las infecciones virales más frecuentes a nivel mundial dada su fácil forma de transmisión, aunado a esto, se ha encontrado su asociación a patologías autoinmunes. Este trabajo nos permitirá conocer el mejor tratamiento y método diagnóstico más adecuado para su resolución clínica favorable. Esta revisión sistemática se elaboró en la base de datos de Literatura Latinoamericana y del Caribe en Ciencias de la Salud, PubMed y de Google Scholar. La búsqueda fue delimitada a estudios realizados en humanos, textos completos, sin importar el idioma de escritura ni restricción de país de procedencia. Se recuperaron un total de 133 artículos en la búsqueda bibliográfica, de los cuales se excluyeron 11 por estar duplicados, 71 artículos fueron descartados por contener breve información o solo se realiza una mención efímera del tema, se prescindieron 15 más en la fase de revisión Full Text, 5 más por no ser estudios en humanos y, por último, 10 opiniones y perspectivas, de los cuales se concluyó que el tratamiento se debe administrar englobando ambas patologías para evitar la exacerbación clínica

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Entorno de clase virtual en Internet

El proyecto descrito en el presente documento consiste en la implementación de un módulo que añade una serie de funcionalidades al proyecto de “e-learning”, previamente existente, denominado . Además de esta parte de implementación, el trabajo consta de un importante apartado documental donde se describen con detalle dos de los estándares más influyentes dentro del campo del aprendizaje virtual: ADL-Scorm y LIP. Los dos objetivos más prioritarios de este proyecto son, por un lado, dar un paso más en la estandarización de los sistemas “e-learning”, aspecto que consideramos de vital importancia para la potenciación de los mismos. Para ello hemos realizado un trabajo de documentación acerca de los dos estándares anteriormente comentados y hemos hecho partícipe al segundo de ellos (LIP) en nuestra implementación. Por otro lado, incorporar la adaptación de los contenidos del curso, es decir, en la forma en que éstos se presentan al alumno dependiendo de sus preferencias y del dispositivo mediante el cual se conectan al sistema. Entendemos que los alumnos son los usuarios que justifican la realización de los sistemas de “e-learning”, por ello, consideramos que este tipo de sistemas no deben sólo basarse en la presentación de contenidos de alta calidad educativa, sino también proporcionar al usuario la posibilidad de poder editar la forma en que estos contenidos son presentados en función de sus preferencias, pero siempre limitados por las características del dispositivo con el cual accedan a dichos contenidos. Además de estos objetivos prioritarios, nuestro proyecto incorpora también mecanismos de seguimiento para facilitar a los tutores su labor de evaluación sobre los alumnos, como son la generación de estadísticas relativas principalmente a la interactuación de los alumnos con el sistema así como un módulo para simplificar la comunicación vía e-mail entre el tutor y sus alumnos. El propósito de este proyecto es que sus contenidos sean incorporados, si no en su totalidad, sí al menos en su gran mayoría, al sistema por razones de “necesidad”. Es decir, que el sistema siga los cauces de estandarización y accesibilidad fijados por sus desarrolladores originales y que éstos se vean reflejados en nuestro proyecto. Esto supondría que hemos alcanzado las metas que al comienzo de este curso académico nos presentó nuestro director de proyecto, el profesor Baltasar Fernández-Manjón y con ello, que este trabajo de Sistemas Informáticos ha supuesto algo más que una labor estrictamente académica. [ABSTRACT] The Project described in this document consists of the implementation of a module which adds several functionalities to the e-learning project, that previously existed, called e-aula. Besides the implementation part, this work has an important documentary section where are described the most influent standards in virtual learning area: ADL-Scorm and LIP. There are two principal objectives in this project: first of all to improve the standardization of e-learning systems, something we consider very significant in order to boost them. As for this, its includes a documentary work on these two standards and also the use of LIP in our implementation. Secondly, we also wanted to adapt the course contents to the preferences of the students when they use them, since we do not consider the high quality contents as the only one aim of e-learning systems, but also to provide the users different options to edit the way these contents appear (obviously this possibility is limited by the device characteristics). Finally, our project tries to make the students evaluations easier, providing stadistics related to the interplay between students and system and making simplier the e-mail communication between tutor and students. The main purpose of this project is to incorporate as many contents as we can to the e-aula project, paying special attention to the standardization and accessibility set by the original developers. In this way we would have achieved the aims which our director, professor Baltasar Fernández Manjón, suggested, and at the same time, this work would have been something more than a simple academic task

Glucagon effect on rat liver protein synthesis in vivo

The in vivo effect of glucagon administration on hepatic polyribosomal profiles has been studied. Glucagon did not change significantly total, free or bound polyribosomal fractions 30-45 minutes after its administration. The combined administration of glucagon plus antiinsulin serum failed to show any significant effect of glucagon over the antiinsulin serum treated control. Glucagon increased valine production in the perfused isolated liver. These results suggest that the well known amino acid catabolic action of glucagon may be preferentially mediated through an increased proteolysis. Since it is known that glucagon increases considerably in vivo the liver cyclic AMP levels then its lack of effect on polyribosomal profiles might indicate that the postulated role for the cyclic nucleotide on liver protein synthesis must be taken cautiously.This work has been supported by a Grant-in-aid from Lilly Indiana S.A. and a research grant (612/g) from Fondo Nacional para el Desarrollo de la Investigación.Peer reviewe

Cellular metabolite distribution and the control of gluconeogenesis in the perfused isolated rat liver

Glucose production was measured in isolated rat livers perfused with 100 ml of blood-free recirculating medium. The gluconeogenic rate using l-alanine as substrate was only 55% of that obtained with l-lactate. The steady-state concentration of gluconeogenic and tricarboxylic acid cycle intermediates were measured in freeze clamped biopsies. Livers perfused with l-lactate displayed higher concentrations of malate, α-glycerophosphate and β-hydroxybutyrate probably as a result of a higher state of reduction of the nicotinamide system. Hexose-phosphate intermediates were also increased when l-lactate was the substrate. Phosphoenolpyruvate and 3-phosphoglycerate were considerably elevated when l-alanine was the glucose precursor. Livers perfused with l-lactate displayed higher cytosolic concentration of all the tricarboxylic acid cycle intermediates except oxaloacetate while glutamate was slightly and aspartare considerably higher when alanine was the substrate. In the mitochondrial compartment the pattern of distribution tended to be the opposite; that is, livers perfused with l-lactate showed lower concentrations of all the intermediates except α-ketoglutarate. The mitochondrial: cytosolic metabolite gradients of all the intermediates whose distribution was studied were higher in livers perfused with l-alanine. The relevance of these findings to the observed differences in the gluconeogenic fluxes are discussed.This work has been supported in part by a Grant-in-aid from Lilly Indiana and a research grant (612/9) from the Spanish Advisory Commission for the Development of Research.Peer reviewe

Control of cell adhesion and migration by Podocalyxin. Implication of Rac1 and Cdc42

23 p.-4 fig.-1 fig.supl.Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell–cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesionThe work was funded with Grants from the Spanish Plan of R&D, SAF2007-61701 and BFU2010-15237Peer reviewe

Cellular redistribution of metabolites during glucagon and insulin control of gluconeogenesis in the isolated perfused rat liver

Livers isolated from fasted rats were perfused in a blood-free recirculating system using alanine (10 mm) as the carbon source. Glucagon at a concentration of 2.1 × 10−9m enhanced gluconeogenesis, ureogenesis, and ketogenesis. The proportion of alanine utilized to glucose formed remained rather constant in all the situations studied, suggesting that the contribution of glycogen breakdown to the total glucose output was negligible. The glucagon stimulation of gluconeogenesis was accompanied by a decrease in the [ATP]/[ADP]ratio and a rise in the reduction state of the cytosolic and mitochondrial NAD systems. The calculation of the intracellular distribution of metabolites indicates that glucagon increases the intramitochondrial oxaloacetate concentration. This finding seems to support the hypothesis of pyruvate carboxylation, the first nonequilibrium enzymic step in the gluconeogenic sequence, as one of the main sites of glucagon action. The rise in the mitochondrial:cytosolic concentration gradient of malate suggests that glucagon may also act by facilitating the transfer of three-carbon units from the mitochondria to the cytosol. The fact that insulin reversed virtually all the glucagon-induced changes strongly suggests that both hormones act on common steps. It is remarkable that these insulin effects occur at glucagon/insulin ratios similar to those normally found in the portal vein of the intact animal.This work has been supported by grants from Lilly Indiana S.A., Fundación Rodríguez Pascual and Comisión Asesora para el Desarrollo de la InvestigaciónPeer reviewe

Altered Ca2+ homeostasis in lymphoblasts from patients with late-onset Alzheimer disease

The authors report calcium (Ca2+) homeostasis features of transformed lymphocytes from patients with late-onset Alzheimer disease and healthy age-matched controls. Alzheimer lymphoblasts show higher basal cytosolic-free [Ca2+] than controls. The antibodies anti-immunoglobulin M or the beta-amyloid (β-amyloid) peptide fragment 25-35-induced elevation of cytosolic-free [Ca2+] was higher in Alzheimer disease lymphoblasts than in control cells. However, the kinetics of Ca2+ replenishment of Ca2+-depleted cells shows a higher accumulation of cytosolic Ca2+ in Alzheimer disease than in control lymphoblasts, which is better appreciated when the Ca2+ efflux is inhibited. Thus, the authors concluded that Alzheimer disease lymphoblasts have a lower Ca2+ buffering capacity than normal cells, probably because of changes in availability or intrinsic functional properties of the intracellular Ca2+-binding structures. Aging alters the kinetics of the Ca2+ replenishment in lymphoblasts in a manner that resembles Alzheimer disease. However, unlike Alzheimer disease, aging does not change the maximum cytosolic-free [Ca2+], suggesting that the mechanisms underlying the altered Ca2+ homeostasis in aging and late-onset Alzheimer disease are different.Peer reviewe

Glucagon and Insulin Control of Gluconeogenesis in the Perfused Isolated Rat Liver. Effects on Cellular Metabolite Distribution

The metabolic effects of glucagon and glucagon plus insulin on the isolated rat livers perfused with 10 mM sodium L‐lactate as substrate were studied. Glucagon stimulated gluconeogenesis, ketogenesis and ureogenesis at the concentration used of 2.1 nM. The addition of insulin to give a glucagon‐to‐insulin ratio of 0.2 reversed all the glucagon effects. The glucagon enhancement of gluconeogenesis was accompanied by a rise in the cytosolic and mitochondrial state of reduction of the NAD system and a fall in the [ATP]/[ADP] ratio. The analysis of the intermediary metabolite concentrations suggested, as possible sites of glucagon action, the steps between pyruvate and phosphoenolpyruvate as well as the reactions catalyzed by phosphofructokinase and/or fructose bisphosphatase. All the changes in metabolite contents were abolished when insulin was present. Glucagon increased the intramitochondrial concentration of all the metabolites, whose intracellular distribution was calculated. The finding of a significant rise in the calculated intramitochondrial concentration of oxaloacetate points to pyruvate carboxylation as an important site of glucagon interaction with the gluconeogenic pathway. A primary event in the glucagon action redistributing intracellular metabolites seems to be the mitochondrial entry of malate. The possibility is discussed that the changes in metabolite cellular distribution were brought about by the increased cellular state of reduction caused by the hormone.This work has been supported by grants from Lilly Indiana S. A.. Fundación Rodríguez Pascual and Comisión Asesora para el Desarrollo de la Investigación.Peer reviewe