Institute Biologia y Genetica Molecular

Valladolid, Spain

Institute Biologia y Genetica Molecular

Valladolid, Spain
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Valdearcos M.,Institute Biologia y Genetica Molecular | Valdearcos M.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | Esquinas E.,Institute Biologia y Genetica Molecular | Esquinas E.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | And 12 more authors.
Journal of Immunology | Year: 2011

The lipins have been described as metabolic enzymes that regulate lipid biosynthesis and also signaling processes by controlling the cellular concentration of bioactive lipids, phosphatidic acid, and diacylgycerol. In the present work we have studied the subcellular localization and role of lipin-1 in human monocyte-derived macrophages. Human macrophages express lipin-1 isoforms α and β. A transfected lipin-1α - enhanced GFP construct associates with membranes of cellular organelles that can be stained with Nile Red. Colocalization experiments with lipid droplet (LD)-specific proteins such as adipophilin/adipose differentiation-related protein/ perilipin 2 or TIP47/perilipin 3 show that both proteins colocalize with lipin-1α in the same cellular structures. Reduction of the expression levels of lipin-1 by small interfering RNA technology does not impair triacylglycerol biosynthesis but reduces the size of LDs formed in response to oleic acid. In agreement with these data, peritoneal macrophages from animals that carry a mutation in the Lpin-1 gene (fld animals) also produce less and smaller LDs in response to oleic acid. Mass spectrometry determinations demonstrate that the fatty acid composition of triacylglycerol in isolated LDs from lipin-1 - deficient cells differs from that of control cells. Moreover, activation of cytosolic group IVA phospholipase A2α, a proinflammatory enzyme that is also involved in LD biogenesis, is also compromised in lipin-1 - deficient cells. Collectively, these data suggest that lipin-1 associates with LDs and regulates the activation of cytosolic group IVA phospholipase A2α in human monocyte-derived macrophages. Copyright © 2011 by The American Association of Immunologists, Inc.

Perez-Cabornero L.,Institute Biologia y Genetica Molecular | Infante M.,Institute Biologia y Genetica Molecular | Velasco E.,Institute Biologia y Genetica Molecular | Lastra E.,Hospital General Yague Of Burgos | And 3 more authors.
International Journal of Colorectal Disease | Year: 2013

Background: Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by heterozygous mutations in mismatch repair (MMR) genes. Approximately 85 % of genetically defined HNPCC patients have germline mutations in MLH1 and MSH2. HNPCC patients are at increased risk of developing extracolonic cancers. The early age of onset, predominantly right-sided colon cancers, and synchronous and metachronous cancers are other features of the syndrome. HNPCC shows heterogeneous clinical phenotypes, and differences in gene mutation frequencies have been observed in some countries. Several investigators have tried to correlate the phenotype with the affected gene. Methods: A total of 46 individuals from 22 unrelated families, of the 264 families fulfilling the inclusion criteria, with deleterious mutations in MLH1, MSH2, or MSH6 genes were identified. We evaluated these clinicopathological features in their relation to different genetic parameters (gene mutated, type of mutation, or alteration of the MMR system in high-risk families) in order to establish a relationship between the phenotype and the genotype in our series. Results: The phenotype of the disease seems not to be influenced by the type of mutation, but rather by the mutated gene. The presence of multiple tumors is associated with mutations in the MSH2 gene. The mean age at diagnosis of the first colorectal cancer (CRC) was almost identical in families with mutations in MLH1 and MSH2, about 50 years of age, but this age may increase by almost 10 years for MSH6 mutation carriers. Conclusion: The identification of genotype-phenotype correlations could provide a more specific surveillance program focused on the individualized risk. © 2013 Springer-Verlag Berlin Heidelberg.

Sanz D.J.,Institute Biologia Y Genetica Molecular | Acedo A.,Institute Biologia Y Genetica Molecular | Infante M.,Institute Biologia Y Genetica Molecular | Duran M.,Institute Biologia Y Genetica Molecular | And 5 more authors.
Clinical Cancer Research | Year: 2010

Purpose: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC). Experimental Design: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells. Results: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC. Conclusions: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations. ©2010 AACR.

Conde S.V.,New University of Lisbon | Nunes Da Silva T.,New University of Lisbon | Gonzalez C.,University of Valladolid | Gonzalez C.,Institute Biologia y Genetica Molecular | And 4 more authors.
British Journal of Nutrition | Year: 2012

We tested the hypothesis that long-term caffeine intake prevents the development of insulin resistance and hypertension in two pathological animal models: the high-fat (HF) and the high-sucrose (HSu) diet rat. We used six groups of animals: control; caffeine-treated (Caff; 1 g/l in drinking water during 15 d); HF; caffeine-treated HF (HFCaff); HSu; caffeine-treated HSu (HSuCaff). Insulin sensitivity was assessed using the insulin tolerance test. Blood pressure, weight gain, visceral fat, hepatic glutathione, plasma caffeine, insulin and NO, and serum NEFA and catecholamines were measured. Caffeine reversed insulin resistance and hypertension induced by both the HF and HSu diets. In the HF-fed animals caffeine treatment restored fasting insulin levels to control values and reversed increased weight gain and visceral fat mass. In the HSu group, caffeine reversed fasting hyperglycaemia and restored NEFA to control values. There were no changes either in plasma NO or in hepatic glutathione levels. In contrast, caffeine totally prevented the increase in serum catecholamines induced by HF and HSu diets. To test the hypothesis that inhibition of the sympathetic nervous system prevents the development of diet-induced insulin resistance we administered carvedilol, an antagonist of β1, β2 and also α1 adrenoceptors, to HF and HSu rats. Carvedilol treatment fully prevented diet-induced insulin resistance and hypertension, mimicking the effect of caffeine. We concluded that long-term caffeine intake prevented the development of insulin resistance and hypertension in HF and HSu models and that this effect was related to a decrease in circulating catecholamines. © 2011 The Authors.

Correnti C.,Fred Hutchinson Cancer Research Center | Clifton M.C.,Fred Hutchinson Cancer Research Center | Abergel R.J.,University of California at Berkeley | Allred B.,University of California at Berkeley | And 6 more authors.
Structure | Year: 2011

Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8 crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities. © 2011 Elsevier Ltd All rights reserved.

Guijas C.,Institute Biologia y Genetica Molecular | Perez-Chacon G.,Institute Biologia y Genetica Molecular | Astudillo A.M.,Institute Biologia y Genetica Molecular | Rubio J.M.,Institute Biologia y Genetica Molecular | And 3 more authors.
Journal of Lipid Research | Year: 2012

Exposure of human peripheral blood monocytes to free arachidonic acid (AA) results in the rapid induction of lipid droplet (LD) formation by these cells. This effect appears specific for AA in that it is not mimicked by other fatty acids, whether saturated or unsaturated. LDs are formed by two different routes: (i) the direct entry of AA into triacylglycerol and (ii) activation of intracellular signaling, leading to increased triacylglycerol and cholesteryl ester formation utilizing fatty acids coming from the de novo biosynthetic route. Both routes can be dissociated by the arachidonyl-CoA synthetase inhibitor triacsin C, which prevents the former but not the latter. LD formation by AA-induced signaling predominates, accounting for 60-70% of total LD formation, and can be completely inhibited by selective inhibition of the group IVA cytosolic phospholipase A2α (cPLA2α), pointing out this enzyme as a key regulator of AA-induced signaling. LD formation in AA-treated monocytes can also be blocked by the combined inhibition of the mitogen-activated protein kinase family members p38 and JNK, which correlates with inhibition of cPLA2α activation by phosphorylation. Collectively, these results suggest that concomitant activation of p38 and JNK by AA cooperate to activate cPLA2α, which is in turn required for LD formation possibly by facilitating biogenesis of this organelle, not by regulating neutral lipid synthesis.

Hidalgo P.I.,University of León | Ullan R.V.,Institute Biotecnologia Of Leon | Albillos S.M.,Institute Biotecnologia Of Leon | Montero O.,Institute Biologia y Genetica Molecular | And 4 more authors.
Fungal Genetics and Biology | Year: 2014

The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H+ antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence. © 2013 Elsevier Inc.

Astudillo A.M.,Institute Biologia y Genetica Molecular | Astudillo A.M.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | Balgoma D.,Institute Biologia y Genetica Molecular | Balgoma D.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | And 4 more authors.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids | Year: 2012

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites. © 2011 Elsevier B.V.

Perez-Chacon G.,Institute Biologia Y Genetica Molecular | Perez-Chacon G.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | Astudillo A.M.,Institute Biologia Y Genetica Molecular | Astudillo A.M.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | And 6 more authors.
Journal of Immunology | Year: 2010

Cellular availability of free arachidonic acid (AA) is an important step in the production of pro- and anti-inflammatory eicosanoids. Control of free AA levels in cells is carried out by the action of phospholipase A2s and lysophospholipid acyltransferases, which are responsible for the reactions of deacylation and incorporation of AA from and into the sn-2 position of phospholipids, respectively. In this work, we have examined the pathways for AA incorporation into phospholipids in human monocytes stimulated by zymosan. Our data show that stimulated cells exhibit an enhanced incorporation of AA into phospholipids that is not secondary to an increased availability of lysophospholipid acceptors due to phospholipase A2 activation but rather reflects the receptor-regulated nature of the AA reacylation pathway. In vitro activity measurements indicate that the receptor-sensitive step of the AA reacylation pathway is the acyltransferase using lysophosphatidylcholine (lysoPC) as acceptor, and inhibition of the enzyme lysoPC acyltransferase 3 by specific small interfering RNA results in inhibition of the stimulated incorporation of AA into phospholipids. Collectively, these results define lysoPC acyltransferase 3 as a novel-signal-regulated enzyme that is centrally implicated in limiting free AA levels in activated cells. Copyright © 2010 by The American Association of Immunologists, Inc.

Casas J.,Institute Biologia y Genetica Molecular | Valdearcos M.,Institute Biologia y Genetica Molecular | Valdearcos M.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas Asociadas | Pindado J.,Institute Biologia y Genetica Molecular | And 4 more authors.
Journal of Lipid Research | Year: 2010

Group IVA cytosolic phospholipase A 2 (cPLA 2 ) plays a role in the microbicidal machinery of immune cells by translocating to phagosomes to initiate the production of antimicrobial eicosanoids. In this work, we have studied the involvement of the cationic cluster of cPLA 2 (Lys 488 / Lys 541 /Lys 543 /Lys 544) in the translocation of the enzyme to the phagosomal cup in human macrophages responding to opsonized zymosan. Phagocytosis was accompanied by an increased mobilization of free arachidonic acid, which was strongly inhibited by pyrrophenone. In transfected cells, a catalytically active enhanced green fl uorescent proteincPLA 2 translocated to the phagocytic cup, which was corroborated by frustrated phagocytosis experiments using immunoglobulin G-coated plates. However, a cPLA 2 mutant in the polybasic cluster that cannot bind the anionic phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP 2) did not translocate to the phagocytic cup. Moreover, an enhanced yellow fl uorescent protein (EYFP)-cPLA 2 and an enhanced cyan fl uorescent protein-pleckstrin homology (PH) domain of the phospholipase C - 1 (PLC - 1) construct that specifi cally recognizes endogenous PIP 2 in the cells both localized at the same sites on the phagosome. High cellular expression of the PH domain inhibited EYFP-cPLA 2 translocation. On the other hand, group V-secreted phospholipase A 2 and group VIA calcium-independent phospholipase A 2 were also studied, but the results indicated that neither of these translocated to the phagosome. Collectively, these data indicate that the polybasic cluster of cPLA 2 (Lys 488 /Lys 541 /Lys 543 /Lys 544) regulates the subcellular localization of the enzyme in intact cells under physiologically relevant conditions. --Casas, J., M. Valdearcos, J. Pindado, J. Balsinde, and M. A. Balboa. The cationic cluster of group IVA phospholipase A2 (Lys488/Lys541/Lys543/ Lys544) is involved in translocation of the enzyme to phagosomes in human macrophages. Copyright © 2010 by the American Society for Biochemistry and Molecular Biology, Inc.

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