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Yuste-Checa P.,Autonomous University of Madrid | Yuste-Checa P.,Institute Investigacion Sanitaria IdiPaZ | Gamez A.,Autonomous University of Madrid | Gamez A.,Institute Investigacion Sanitaria IdiPaZ | And 10 more authors.
Human Mutation | Year: 2015

Congenital disorder of glycosylation type Ia (PMM2-CDG), the most common form of CDG, is caused by mutations in the PMM2 gene that reduce phosphomannomutase 2 (PMM2) activity. No curative treatment is available. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The effects of the mutations on PMM2/Pmm2 stability, oligomerization, and enzyme activity were explored in an optimized bacterial system. The mutant proteins were associated with an enzymatic activity of up to 47.3% as compared with wild type (WT). Stability analysis performed using differential scanning fluorimetry and a bacterial transcription-translation-coupled system allowed the identification of several destabilizing mutations (p.V44A, p.D65Y, p.R123Q, p.R141H, p.R162W, p.F207S, p.T237M, p.C241S). Exclusion chromatography identified one mutation, p.P113L, that affected dimer interaction. Expression analysis of the p.V44A, p.D65Y, p.R162W, and p.T237M mutations in a eukaryotic expression system under permissive folding conditions showed the possibility of recovering their associated PMM2 activity. Together, the results suggest that some loss-of-function mutations detected in PMM2-CDG patients could be destabilizing, and therefore PMM2 activity could be, in certain cases, rescuable via the use of synergetic proteostasis modulators and/or chaperones. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The results suggest that PMM2-CDG could be considered a conformational disease and therefore PMM2 activity could be rescuable via the use of small stabilizer molecules. Unfortunately, this mouse model is inadequate for testing that kind of compounds since neither mutation causes conformational instability. © 2015 WILEY PERIODICALS, INC..


Husi H.,University of Glasgow | Sanchez-Nino M.D.,Institute Investigacion Sanitaria IDIPAZ | Delles C.,University of Glasgow | Mullen W.,University of Glasgow | And 5 more authors.
BMC Systems Biology | Year: 2013

Background: Acute kidney injury (AKI) is a frequent condition in hospitalised patients undergoing major surgery or the critically ill and is associated with increased mortality. Based on the volume of the published literature addressing this condition, reporting both supporting as well as conflicting molecular evidence, it is apparent that a comprehensive analysis strategy is required to understand and fully delineate molecular events and pathways which can be used to describe disease induction and progression as well as lead to a more targeted approach in intervention therapies.Results: We used a Systems Biology approach coupled with a de-novo high-resolution proteomic analysis of kidney cortex samples from a mouse model of folic acid-induced AKI (12 animals in total) and show comprehensive mapping of signalling cascades, gene activation events and metabolite interference by mapping high-resolution proteomic datasets onto a de-novo hypothesis-free dataspace. The findings support the involvement of the glutamatergic signalling system in AKI, induced by over-activation of the N-methyl-D-aspartate (NMDA)-receptor leading to apoptosis and necrosis by Ca2+-influx, calpain and caspase activation, and co-occurring reactive oxygen species (ROS) production to DNA fragmentation and NAD-rundown. The specific over-activation of the NMDA receptor may be triggered by the p53-induced protein kinase Dapk1, which is a known non-reversible cell death inducer in a neurological context. The pathway mapping is consistent with the involvement of the Renin-Angiotensin Aldosterone System (RAAS), corticoid and TNFα signalling, leading to ROS production and gene activation through NFκB, PPARγ, SMAD and HIF1α trans-activation, as well as p53 signalling cascade activation. Key elements of the RAAS-glutamatergic axis were assembled as a novel hypothetical pathway and validated by immunohistochemistry.Conclusions: This study shows to our knowledge for the first time in a molecular signal transduction pathway map how AKI is induced, progresses through specific signalling cascades that may lead to end-effects such as apoptosis and necrosis by uncoupling of the NMDA receptor. Our results can potentially pave the way for a targeted pharmacological intervention in disease progression or induction. © 2013 Husi et al.; licensee BioMed Central Ltd.


Blanco-Rivero J.,Autonomous University of Madrid | Blanco-Rivero J.,Institute Investigacion Sanitaria IdIPaz | Sastre E.,Autonomous University of Madrid | Caracuel L.,Autonomous University of Madrid | And 3 more authors.
PLoS ONE | Year: 2013

Objectives: The aim of this study was to investigate in rat mesenteric artery whether breast feeding (BF) affects the vasomotor response induced by electrical field stimulation (EFS), participation by different innervations in the EFS-induced response and the mechanism/s underlying these possible modifications. Methods: Experiments were performed in female Sprague-Dawley rats (3 months old), divided into three groups: Control (in oestrous phase), mothers after 21 days of BF, and mothers that had recovered their oestral cycle (After BF, in oestrous phase). Vasomotor response to EFS, noradrenaline (NA) and nitric oxide (NO) donor DEA-NO were studied. Neuronal NO synthase (nNOS) and phosphorylated nNOS (P-nNOS) protein expression were analysed and NO, superoxide anion (O2 .-), NA and ATP releases were also determined. Results: EFS-induced contraction was higher in the BF group, and was recovered after BF. 1 μmol/L phentolamine decreased the response to EFS similarly in control and BF rats. NA vasoconstriction and release were similar in both experimental groups. ATP release was higher in segments from BF rats. 0.1 mmol/L L-NAME increased the response to EFS in both control and BF rats, but more so in control animals. BF decreased NO release and did not modify O2 .- production. Vasodilator response to DEA-NO was similar in both groups, while nNOS and P-nNOS expressions were decreased in segments from BF animals. Conclusion: Breast feeding increases EFS-induced contraction in mesenteric arteries, mainly through the decrease of neuronal NO release mediated by decreased nNOS and P-nNOS expression. Sympathetic function is increased through the increased ATP release in BF rats. © 2013 Blanco-Rivero et al.


Sastre E.,Autonomous University of Madrid | Balfagon G.,Autonomous University of Madrid | Balfagon G.,Institute Investigacion Sanitaria IdIPaz | Revuelta-Lopez E.,Complutense University of Madrid | And 5 more authors.
Clinical Science | Year: 2012

In the present study, we analysed possible alterations in adrenergic, nitrergic and sensory functioning in mesenteric arteries from rats at 1 and 21 months after partial portal vein ligation, and the mechanisms involved in these alterations, if any. For this purpose, we analysed the vasoconstrictor response to EFS (electrical field stimulation) and the effect of the α-antagonist phentolamine, the NOS (nitric oxide synthase) inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and the CGRP (calcitonin gene-related peptide) receptor antagonist CGRP-(8-37) in mesenteric segments from ST (short-term; 1 month) and LT (long-term; 21 months) SO (shamoperated) and pre-hepatic PH (portal hypertensive) rats. The vasomotor responses to NA (noradrenaline), the NO donor DEA-NO (diethylamine NONOate) and CGRP were analysed. NA, NO and CGRP releases were measured. Phospho-nNOS (neuronal NOS) expression was studied. The vasoconstrictor response to EFS was decreased in STPH animals. Phentolamine decreased this vasoconstrictor response more strongly in SO animals. Both L-NAME and CGRP-(8-37) increased vasoconstrictor response to EFS more strongly in PH than SO segments. PH did not modify vasomotor responses to NA, DEA-NO or CGRP, but it decreased NA release while increasing those of NO and CGRP. Phospho-nNOS expression was increased by PH. In LTPH, no differences were observed in vasoconstrictor response to EFS, vasomotor responses or neurotransmitter release when compared with age-matched SO animals. In conclusion, the mesenteric innervation may participate in the development of the characteristic hyperdynamic circulation observed in STPH through the joint action of decreased adrenergic influence, and increased nitrergic and sensory innervations influences. The participation of each innervation normalizes under conditions of LTPH. © The Authors Journal compilation © 2012 Biochemical Society.


Del Campo L.,University of Amsterdam | Del Campo L.,Autonomous University of Madrid | Del Campo L.,Institute Investigacion Sanitaria IdiPAZ | Tuna B.G.,University of Amsterdam | And 4 more authors.
Clinical Science | Year: 2013

Increasing evidence shows that sex hormones exert a protective effect on the vasculature, especially in the regulation of the active vasomotor responses. However, whether sex hormones affect vascular remodelling is currently unclear. In the present study, we tested the hypothesis that testosterone in males and β-oestradiol in females prevent inward remodelling, possibly through inhibition of cross-linking activity induced by enzymes of the TG (transglutaminase) family. Small mesenteric arteries were isolated from male and female Wistar rats. Dose-dependent relaxation to testosterone and β-oestradiol was inhibited by the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester), confirming that these hormones induce NO release. When arteries were cannulated, pressurized and kept in organ culture with ET-1 (endothelin-1) for 3 days we observed strong vasoconstriction and inward remodelling. Remodelling was significantly inhibited by testosterone in males, and by β-oestradiol in females. This preventive effect of sex hormones was not observed in the presence of L-NAME. Inward remodelling was also reduced by the inhibitor of TG L682.777, both in males and females. In arteries from female rats, ET-1 increased TG activity, and this effect was prevented by β-oestradiol. L-NAME induced a significant increase in TG activity in the presence of sex hormones in arteries from both genders. We conclude thattestosterone and β-oestradiol prevent constriction-induced inward remodelling. Inward remodelling, both in males and females, depends on NO and TG activity. In females, inhibition of inward remodelling could be mediated by NO-mediated inhibition of TG activity. © The Authors Journal compilation. © 2013 Biochemical Society.


Pifl C.,Medical University of Vienna | Hornykiewicz O.,Medical University of Vienna | Blesa J.,University of Navarra | Blesa J.,Charles III University of Madrid | And 6 more authors.
Journal of Neurochemistry | Year: 2013

We recently found severe noradrenaline deficits throughout the thalamus of patients with Parkinson's disease [C. Pifl, S. J. Kish and O. Hornykiewicz Mov Disord. 27, 2012, 1618.]. As this noradrenaline loss was especially severe in nuclei of the motor thalamus normally transmitting basal ganglia motor output to the cortex, we hypothesized that this noradrenaline loss aggravates the motor disorder of Parkinson's disease. Here, we analysed noradrenaline, dopamine and serotonin in motor (ventrolateral and ventroanterior) and non-motor (mediodorsal, centromedian, ventroposterior lateral and reticular) thalamic nuclei in MPTP-treated monkeys who were always asymptomatic; who recovered from mild parkinsonism; and monkeys with stable, either moderate or severe parkinsonism. We found that only the symptomatic parkinsonian animals had significant noradrenaline losses specifically in the motor thalamus, with the ventroanterior motor nucleus being affected only in the severe parkinsonian animals. In contrast, the striatal dopamine loss was identical in both the mild and severe symptom groups. MPTP-treatment had no significant effect on noradrenaline in non-motor thalamic nuclei or dopamine and serotonin in any thalamic subregion. We conclude that in the MPTP primate model, loss of noradrenaline in the motor thalamus may also contribute to the clinical expression of the parkinsonian motor disorder, corroborating experimentally our hypothesis on the role of thalamic noradrenaline deficit in Parkinson's disease. Parkinson patients and animals with experimentally reduced thalamic noradrenaline exhibit pathological thalamic discharge patterns. In Parkinson's disease, motor thalamic noradrenaline is profoundly reduced. MPTP-induced parkinsonian monkeys had in addition to basal ganglia dopamine loss noradrenaline losses specifically in the motor thalamus, with the ventroanterior motor nucleus being affected only in severe parkinsonism. Thus, noradrenaline, specifically in the thalamus, appears to be an important player in the pathophysiology of Parkinson's disease. Read the Editorial Highlight for this article on doi: 10.1111/jnc.12167. © 2013 International Society for Neurochemistry.


Garcia-Rodriguez B.,Spanish University for Distance Education (UNED) | Garcia-Rodriguez B.,Institute Investigacion Sanitaria IdiPaz | Casares-Guillen C.T.,Spanish University for Distance Education (UNED) | Molina J.A.,Hospital 12 Of Octubre | And 4 more authors.
Revista de Neurologia | Year: 2011

Introduction. Previous research has shown that correct identification of emotional facial expressions (EFE) depends on the cognitive resources that are available. In this study, we examine whether the capacity to identify EFE in a dual task paradigm is affected in Parkinson's disease (PD). Aim. To investigate the interference generated by introducing a secondary task in EFE processing during the encoding and recovery of the facial expression in non-medicated PD patients. Subjects and methods. A total of 14 patients with de novo PD and 28 healthy adults identified 24 EFE under two conditions: simultaneous encoding along with a secondary task and introduction of the secondary task between the time that spans the encoding of the primary task and the response time latency. Results. Results showed that identification of EFE by patients with PD was significantly worse than by healthy adults in the simultaneous encoding condition. In contrast, no differences were found when the interference of the secondary task took place in the phase involving recovery of information of the primary task. Conclusions. Patients with PD only display specific deficits in processing EFE when the task consumes high levels of the resources required for divided attention, as occurs in everyday situations. © 2011 Revista de Neurología.


de la Cueva A.,Institute Investigaciones Biomedicas | de la Cueva A.,IMDEA Madrid Institute for Advanced Studies | Ramirez de Molina A.,Institute Investigaciones Biomedicas | Ramirez de Molina A.,IMDEA Madrid Institute for Advanced Studies | And 8 more authors.
PLoS ONE | Year: 2013

Background:Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.Methodology/Principal Findings:ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Conclusion/Significance:Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors. © 2013 de la Cueva et al.


Ramirez de Molina A.,IMDEA Madrid Institute for Advanced Studies | Ramirez de Molina A.,Institute Investigacion Sanitaria IdiPAZ | de la Cueva A.,Institute Investigacion Sanitaria IdiPAZ | Machado-Pinilla R.,Institute Investigaciones Biomedicas | And 6 more authors.
Current Cancer Drug Targets | Year: 2012

We have analyzed the response of primary cultures derived from tumor specimens of non small cell lung cancer (NSCLC) patients to choline kinase α (ChoKα) inhibitors. ChoKα inhibitors have been demonstrated to increase ceramides levels specifically in tumor cells, and this increase has been suggested as the mechanism that explain its proapoptotic effect in cancer cells. Here, we have investigated the molecular mechanism associated to the intrinsic resistance, and found that other enzyme involved in lipid metabolism, acid ceramidase (ASAH1), is specifically upregulated in resistant tumors. NSCLC cells with acquired resistance to ChoKα inhibitors also display increased levels of ASAH1. Accordingly, ASAH1 inhibition synergistically sensitizes lung cancer cells to the antiproliferative effect of ChoKα inhibitors. Thus, the determination of the levels of ASAH1 predicts sensitivity to targeted therapy based on ChoKα specific inhibition and represents a model for combinatorial treatments of ChoKα inhibitors and ASAH1 inhibitors. Considering that ChoKα inhibitors have been recently approved to enter Phase I clinical trials by the Food and Drug Administration (FDA), these findings are anticipating critical information to improve the clinical outcome of this family of novel anticancer drugs under development. © 2012 Bentham Science Publishers.


PubMed | Institute Investigacion Sanitaria IdiPAZ
Type: Journal Article | Journal: Current cancer drug targets | Year: 2012

We have analyzed the response of primary cultures derived from tumor specimens of non small cell lung cancer (NSCLC) patients to choline kinase (ChoK) inhibitors. ChoK inhibitors have been demonstrated to increase ceramides levels specifically in tumor cells, and this increase has been suggested as the mechanism that explain its proapoptotic effect in cancer cells. Here, we have investigated the molecular mechanism associated to the intrinsic resistance, and found that other enzyme involved in lipid metabolism, acid ceramidase (ASAH1), is specifically upregulated in resistant tumors. NSCLC cells with acquired resistance to ChoK inhibitors also display increased levels of ASAH1. Accordingly, ASAH1 inhibition synergistically sensitizes lung cancer cells to the antiproliferative effect of ChoK inhibitors. Thus, the determination of the levels of ASAH1 predicts sensitivity to targeted therapy based on ChoK specific inhibition and represents a model for combinatorial treatments of ChoK inhibitors and ASAH1 inhibitors. Considering that ChoK inhibitors have been recently approved to enter Phase I clinical trials by the Food and Drug Administration (FDA), these findings are anticipating critical information to improve the clinical outcome of this family of novel anticancer drugs under development.

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