A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

Measurements of W H and ZH production in the H → bb¯ decay channel in pp collisions at 13 TeV with the ATLAS detector

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UA

The Ras family of GTPases in cancer cell invasion

The ability of tumoral cells to invade surrounding tissues is a prerequisite for metastasis. This is the most life-threatening event of tumor progression, and so research is intensely focused on elucidating the mechanisms responsible for invasion and metastasis. The Ras superfamily of GTPases comprises several subfamilies of small GTP-binding proteins whose functions include the control of proliferation, differentiation, and apoptosis, as well as cytoskeleton organization. The development of metastasis is a multistep process that requires coordinated activation of proliferation, motility, changes in normal cell-to-cell and cell-to-substrate contacts, degradation of extracellular matrix, inhibition of apoptosis, and adaptation to an inappropriate tissue environment. Several members of the Ras superfamily of proteins have been implicated in these processes. The present review summarizes the current knowledge in this field.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41830/1/18-57-1-65_00570065.pd

A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

<p>Abstract</p> <p>Background</p> <p>Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.</p> <p>Results</p> <p>Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the β domain of VHL and a hydroxyproline-containing HIFα peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFα peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.</p> <p>Conclusion</p> <p>The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.</p

The effect of paricalcitolon dialysate protein loss in peritoneal dialysis patients

Ever since peritoneal dialysis (PD) has been used in the treatment of chronic kidney disease (CKD), high peritoneal protein loss has been observed on each PD exchange. In adult patients, the loss has been estimated at 6 to 13 g daily. Paricalcitol, a selective activator of vitamin D receptors (VDR), is successfully used as a treatment of hyperparathyroidism secondary to CKD. In addition, it has been proposed for reducing proteinuria in patients with CKD. Nonetheless, little is known about its effect on peritoneal protein loss (PPL) in patients on PD, namely after the identification of VDRon the peritoneal membrane. The aim of this study wasto examine the effect of paricalcitol on PPL in PD patients

Non-invasive monitoring of hypoxia-inducible factor activation by optical imaging during antiangiogenic treatment in a xenograft model of ovarian carcinoma

Open Access Article.Targeting the hypoxia response pathway and angiogenesis are two promising therapeutic strategies for cancer treatment. Their use as single strategies has important limitations. Thus, development of combined regimens has become an important step toward improving therapeutic efficacy. Also, non-invasive monitoring of the response to targeted biological therapies, as well as determination of the optimal schedule for combination regimens has become an active field of research over the last five years, with relevance for both preclinical and clinical settings. Here, we used an optical imaging method to non-invasively monitor the functional changes in HIF activity in response to antiangiogenic treatment in a xenograft model of human ovarian carcinoma. A bioluminescent reporter construct containing nine copies of the hypoxia response element upstream of the luciferase gene (9xHRE-luciferase) was characterized in vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft model of ovarian carcinoma by means of optical imaging. We showed that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the HIF functional reporter occurs before the onset of massive tumor growth. However, this system failed to detect hypoxia induced upon antiangiogenic treatment due to the compensating effects of increased hypoxia and decreased tumor cell viability caused by imbalanced neovascularization vs. tumor expansion. Therefore, the readout based on HIF functional reporter could be conditioned by the dynamics of tumor growth and angiogenesis, which is highly variable depending on the tumor type, tumor model and stage of progression.This study was supported by grants from the Ministerio de Ciencia y Tecnología/Ministerio de Ciencia e Innovación (SAF2008-03147 to LdP and SAF2010-19256 to BJ), Comunidad Autónoma de Madrid (S-SAL-0311_2006) and the 7th Research Framework Programme of the European Union (METOXIA, project ref. HEALTH-F2-2009-222741). B.M.P. and V.G. have been supported by a grant from the Comunidad Autónoma de Madrid (S-SAL-0311_2006).Peer Reviewe

Measurement of hadronic event shapes in high-pT multijet final states at √s = 13 TeV with the ATLAS detecto

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UA

Search for dark matter produced in association with a dark higgs boson decaying into W ± W∓ or ZZ in fully hadronic final states from √s= 13 TeV pp Collisions Recorded with the ATLAS Detector

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UA

The ZEUS Forward Plug Calorimeter with Lead-Scintillator Plates and WLS Fiber Readout

A Forward Plug Calorimeter (FPC) for the ZEUS detector at HERA has been built as a shashlik lead-scintillator calorimeter with wave length shifter fiber readout. Before installation it was tested and calibrated using the X5 test beam facility of the SPS accelerator at CERN. Electron, muon and pion beams in the momentum range of 10 to 100 GeV/c were used. Results of these measurements are presented as well as a calibration monitoring system based on a $^{60}$Co source.Comment: 38 pages (Latex); 26 figures (ps

Measurements of top-quark pair single- and double-differential cross-sections in the all-hadronic channel in pp collisions at √s = 13 TeV using the ATLAS detector

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UA