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BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has infected several animal species, including dogs, presumably via human-to-animal transmission. Most infected dogs reported were asymptomatic, with low viral loads. However, in this case we detected SARS-CoV-2 in a dog from the North African coastal Spanish city of Ceuta presenting hemorrhagic diarrhea, a disease also reported earlier on in an infected dog from the USA. CASE PRESENTATION: In early January 2021, a West Highland Terrier pet dog from Ceuta (Spain) presented hemorrhagic diarrhea with negative tests for candidate microbial pathogens. Since the animal was in a household whose members suffered SARS-CoV-2 in December 2020, dog feces were analyzed for SARS-CoV-2, proving positive in a two-tube RT-PCR test, with confirmation by sequencing a 399-nucleotide region of the spike (S) gene. Furthermore, next-generation sequencing (NGS) covered > 90% SARS-CoV-2 genome sequence, allowing to classify it as variant B.1.177. Remarkably, the sequence revealed the Ile402Val substitution in the spike protein (S), of potential concern because it mapped in the receptor binding domain (RBD) that mediates virus interaction with the cell. NGS reads mapping to bacterial genomes showed that the dog fecal microbiome fitted best the characteristic microbiome of dog's acute hemorrhagic diarrhea. CONCLUSION: Our findings exemplify dog infection stemming from the human SARS-CoV-2 pandemic, providing nearly complete-genome sequencing of the virus, which is recognized as belonging to the B.1.177 variant, adding knowledge on variant circulation in a geographic region and period for which there was little viral variant characterization. A single amino acid substitution found in the S protein that could have been of concern is excluded to belong to this category given its rarity and intrinsic nature. The dog's pathology suggests that SARS-CoV-2 could affect the gastrointestinal tract of the dog.
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COVID-19 , Enfermedades de los Perros , Animales , COVID-19/veterinaria , Diarrea/veterinaria , Enfermedades de los Perros/diagnóstico , Perros , Humanos , Nucleótidos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
BACKGROUND: The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS: We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS: The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS: EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.
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Variaciones en el Número de Copia de ADN , Reacción en Cadena de la Polimerasa/métodos , Receptores de LDL/genética , Sondas de ADN/química , Sondas de ADN/metabolismo , Colorantes Fluorescentes/química , Humanos , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa Multiplex , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Maternal smoking during pregnancy is a major risk factor for adverse health outcomes. The main objective of the study was to assess the impact of in utero tobacco exposure on DNA methylation in children born at term with appropriate weight at birth. METHODS: Twenty mother-newborn dyads, after uncomplicated pregnancies, in the absence of perinatal illness were included. All mothers were healthy with no cardiovascular risk factors, except for the associated risks among those mothers who smoked. Umbilical cord blood and maternal peripheral venous blood were collected and an epigenome-wide association study was performed using a 450 K epigenome-wide scan (Illumina Infinium HumanMethylation 450BeadChip) with adjustment to normalize the DNA methylation for data cell variability in whole blood. RESULTS: The maternal plasmatic cotinine levels ranged from 10.70-115.40 ng/ml in the exposed group to 0-0.59 ng/ml in the non-exposed group. After adjusting for multiple comparisons in 427102 probes, statistically significant differences for 31 CpG sites, associated to 25 genes were observed. There was a greater than expected proportion of statistically-significant loci located in CpG islands (Fisher's exact test, p = 0.029) and of those CpG islands, 90.3% exhibit higher methylation levels in the exposed group. The most striking and significant CpG site, cg05727225, is located in the chromosome 11p15.4, within the adrenomedullin gene. CONCLUSIONS: In utero tobacco exposure, even in the absence of fetal growth restriction, may alter the epigenome, contributing to global DNA hypomethylation. Therefore, DNA status can be used as a biomarker of prenatal insults. Considering the possibility to reverse epigenetic modifications, a window of opportunity exists to change the programmed chronic disease.
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Metilación de ADN , Exposición Materna , Contaminación por Humo de Tabaco , Adrenomedulina/química , Adulto , Análisis por Conglomerados , Islas de CpG , Epigénesis Genética , Epigenómica , Femenino , Sangre Fetal , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Inmunoensayo , Recién Nacido , Madres , Embarazo , Factores de Riesgo , Nicotiana/efectos adversos , Adulto JovenRESUMEN
BACKGROUND: Abdominal obesity (AO) is a common modifiable risk factor for certain non-communicable diseases associated with enhanced oxidative stress (OS). The objective of this work was to investigate whether the interaction between antioxidant vitamin intake and OS-related polymorphisms modulates gene-associated anthropometry in a Spanish population. METHODS: A total of 246 subjects with AO, and 492 age and gender matched non-AO subjects were included in the study. Anthropometric, biochemical, and OS parameters, and antioxidant dietary intake data were assessed using validated procedures. DNA from white blood cells was isolated and the genotype of seven polymorphisms from genes involved in OS (pro-oxidant and antioxidant) were analyzed using the SNPlex system. The effects of the c.-793T > C polymorphism on promoter activity and thus thioredoxin (TXN) activity were examined using reporter assays. RESULTS: The AO group had higher 8-Oxo-2'-deoxyguanosine levels and took in less vitamin A and vitamin E compared to the non-AO group. Logistic regression analysis revealed that the rs2301241 polymorphism in TXN and rs740603 in catechol-O-methyltransferase (COMT) were associated with waist circumference (WC) and AO. Moreover, these polymorphisms were more strongly associated with variations in WC in subjects with low vitamin E intakes. A promoter assay revealed that the T to C conversion at c.-793 (rs2301241) induced a more than two fold increase in reporter gene expression. CONCLUSIONS: WC is associated both with dietary vitamin E intake and genetic variants of TXN and COMT suggesting that existence of a complex nutrigenetic pathway that involves regulation of AO.
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Catecol O-Metiltransferasa/genética , Nutrigenómica , Obesidad/genética , Tiorredoxinas/genética , Vitamina E/química , Circunferencia de la Cintura , Adulto , Factores de Edad , Anciano , Antropometría , Antioxidantes/química , Estudios de Casos y Controles , Dieta , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Factores Sexuales , España , Tiorredoxinas/químicaRESUMEN
RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics, RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G1, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G1/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27 levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240 of RhoE as the Skp2-interacting domain and Lys(235) as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation of RhoE by Skp2 regulates its protein levels to control cellular proliferation.
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Fase G1/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Ubiquitinación/fisiología , Proteínas de Unión al GTP rho/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas de Unión al GTP rho/genéticaRESUMEN
Copy number variations (CNVs) are a type of genetic variation involving from 50 base pairs (bps) to millions of bps and, in a general point of view, can include alterations of complete chromosomes. As CNVs mean the gain or loss of DNA sequences, their detection requires specific techniques and analysis. We have developed Easy One-Step Amplification and Labeling for CNV Detection (EOSAL-CNV) by fragment analysis in a DNA sequencer. The procedure is based on a single PCR reaction for amplification and labeling of all fragments included. The protocol includes specific primers for the amplification of the regions of interest with a tail in each of the primers (one for forward and another for the reverse primers) together with primers for tail amplification. One of the primers for tail amplification is labeled with a fluorophore, allowing the amplification and labeling in the same reaction. Combination of several tail pairs and labels allows the detection of DNA fragment by different fluorophores and increases the number of fragments that can be analyzed in one reaction. PCR products can be analyzed without any purification on a DNA sequencer for fragment detection and quantification. Finally, simple and easy calculations allow the detection of fragments with deletions or extra copies. The use of EOSAL-CNV allows simplifying and reducing costs in sample analysis for CNV detection.
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Variaciones en el Número de Copia de ADN , Reacción en Cadena de la Polimerasa/métodosRESUMEN
BACKGROUND: Low birth weight has been linked to an increased risk to develop obesity, type 2 diabetes, and hypertension in adult life, although the mechanisms underlying the association are not well understood. The objective was to determine whether the metabolomic profile of plasma from umbilical cord differs between low and normal birth weight newborns. METHODS: Fifty healthy pregnant women and their infants were selected. The eligibility criteria were being born at term and having a normal pregnancy. Pairs were grouped according to their birth weight: low birth weight (LBW, birth weight < 10th percentile, n = 20) and control (control, birth weight between the 75th-90th percentiles, n = 30). Nuclear Magnetic Resonance (NMR) was used to generate metabolic fingerprints of umbilical cord plasma samples. Simultaneously, the metabolomic profiles of the mothers were analysed. The resulting data were subjected to chemometric, principal component and partial least squares discriminant analyses. RESULTS: Umbilical cord plasma from LBW and control newborns displayed a clearly differentiated metabolic profile. Seven metabolites were identified that discriminate the LBW from the control group. LBW newborns had lower levels of choline, proline, glutamine, alanine and glucose than did the control newborns, while plasma levels of phenylalanine and citrulline were higher in LBW newborns (p < 0.05). No significant differences were found between the two groups of mothers. CONCLUSIONS: Low birth weight newborns display a differential metabolomic profile than those of normal birth weight, a finding not present in the mothers. The meaning and the potential utility of the findings as biomarkers of risk need to be addressed in future studies.
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Recién Nacido de Bajo Peso/sangre , Recién Nacido de Bajo Peso/metabolismo , Metabolómica , Cordón Umbilical/metabolismo , Adulto , Estudios de Casos y Controles , Análisis Discriminante , Femenino , Humanos , Recién Nacido , Análisis de los Mínimos Cuadrados , Madres , EmbarazoRESUMEN
Patients with high cholesterol and glucose levels are at high risk for cardiovascular disease. The Sterol Regulatory Element Binding Protein (SREBP) system regulates genes involved in lipid, cholesterol and glucose pathways. Autosomal Dominant Hypercholesterolemias (ADHs) are a group of diseases with increased cholesterol levels. They affect 1 out of every 500 individuals. About 20-30% of patients do not present any mutation in the known genes (LDLR, APOB and PCSK9). ADHs constitute a good model to identify the genes involved in the alteration of lipid levels or possible therapeutic targets. In this paper, we studied whether a mutation in the SREBP system could be responsible for ADH and other metabolic alterations present in these patients. Forty-one ADH patients without mutations in the main responsible genes were screened by direct sequencing of SREBP system genes. A luciferase reporter assay of the found mutation and an oral glucose tolerance test in carriers and non-carriers were performed. We found a novel mutation in the SREBF2 gene that increases transcription levels and cosegregates with hypercholesterolemia, and we found increased glucose levels in one family. SREBP2 is known to be involved in cholesterol synthesis, plasma levels and glucose metabolism in humans. The found mutation may involve the SREBF2 gene in hypercholesterolemia combined with hyperglycemia.
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UNLABELLED: There are no effective antifibrotic therapies for patients with liver diseases. We performed an experimental and translational study to investigate whether ghrelin, an orexigenic hormone with pleiotropic properties, modulates liver fibrogenesis. Recombinant ghrelin was administered to rats with chronic (bile duct ligation) and acute (carbon tetrachloride) liver injury. Hepatic gene expression was analyzed by way of microarray analysis and quantitative polymerase chain reaction. The hepatic response to chronic injury was also evaluated in wild-type and ghrelin-deficient mice. Primary human hepatic stellate cells were used to study the effects of ghrelin in vitro. Ghrelin hepatic gene expression and serum levels were assessed in patients with chronic liver diseases. Ghrelin gene polymorphisms were analyzed in patients with chronic hepatitis C. Recombinant ghrelin treatment reduced the fibrogenic response, decreased liver injury and myofibroblast accumulation, and attenuated the altered gene expression profile in bile duct-ligated rats. Moreover, ghrelin reduced the fibrogenic properties of hepatic stellate cells. Ghrelin also protected rats from acute liver injury and reduced the extent of oxidative stress and inflammation. Ghrelin-deficient mice developed exacerbated hepatic fibrosis and liver damage after chronic injury. In patients with chronic liver diseases, ghrelin serum levels decreased in those with advanced fibrosis, and ghrelin gene hepatic expression correlated with expression of fibrogenic genes. In patients with chronic hepatitis C, polymorphisms of the ghrelin gene (-994CT and -604GA) influenced the progression of liver fibrosis. CONCLUSION: Ghrelin exerts antifibrotic effects in the liver and may represent a novel antifibrotic therapy.
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Ghrelina/fisiología , Ghrelina/uso terapéutico , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/prevención & control , Animales , Células Cultivadas , Progresión de la Enfermedad , Humanos , Masculino , Ratones , Ratas , Ratas Wistar , Proteínas Recombinantes/uso terapéuticoRESUMEN
CONTEXT: Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by increased low-density lipoprotein (LDL)-cholesterol levels, leading to high risk of premature cardiovascular disease. More than 900 mutations in LDL receptor, six in APOB and 10 in PCSK9 have been identified as a cause of the disease in different populations. All known mutations in PCSK9 causing hypercholesterolemia produce an increase in the enzymatic activity of this protease. Up to now, there are data about the implication of PCSK9 in ADH in a low number of populations, not including a Spanish population. OBJECTIVE: The objective of the study was to study the prevalence of PCSK9 mutations in ADH Spanish population. PARTICIPANTS: We screened PCSK9 gene in 42 independent ADH patients in whom mutations in LDL receptor and APOB genes had been excluded. RESULTS: None of the known mutations causing ADH was detected in our sample, but we found two variations in the promoter region that could cause ADH, c.-288G>A and c.-332C>A (each in one proband). The analysis of the effect of these two variations on the transcription activity of the PCSK9 promoter showed that c.-288G>A did not modify the transcription, whereas c.-332C>A variant caused a 2.5-fold increase when compared with the wild-type sequence, either with or without lovastatin. CONCLUSIONS: PCSK9 is a rare cause of ADH in Spanish population and, up to what we know, none of the previously described mutations has been detected. We have identified a new mutation that could cause ADH by increasing the transcription of PCSK9.
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Hiperlipoproteinemia Tipo II/genética , Regiones Promotoras Genéticas , Serina Endopeptidasas/genética , Adulto , Animales , Secuencia de Bases , Estudios de Casos y Controles , Células Cultivadas , Expresión Génica , Frecuencia de los Genes , Humanos , Ratones , Persona de Mediana Edad , Proteínas Mutantes/genética , Células 3T3 NIH , Polimorfismo de Nucleótido Simple , Proproteína Convertasa 9 , Proproteína Convertasas , Serina Endopeptidasas/sangre , España , TransfecciónRESUMEN
Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G(1) cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27(Kip1) and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27(Kip1) at the level of transcription. We further show that BCR-ABL also regulates p27(Kip1) protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27(Kip1) by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27(Kip1). We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27(Kip1) mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27(Kip1) contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27(Kip1) and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias.
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Proteínas Portadoras/metabolismo , Proteínas de Fusión bcr-abl/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas Asociadas a Fase-S/biosíntesis , Benzamidas , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Fosforilación , Piperazinas , Pirimidinas/farmacología , Proteína de Retinoblastoma/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is associated with higher levels of inflammatory mediators such as chemokines, which contribute to an increased risk of premature atherosclerosis in these patients. We studied the response of chemokines related to early atherosclerotic processes during an oral unsaturated fat load test (OFLT) in patients with heterozygous FH and compared this response to normolipidemic and normoglycemic subjects. METHODS: Blood samples were taken from 12 FH patients and 20 healthy controls with a similar age, gender distribution, and body mass index. Plasma chemokine levels were determined in both groups in a fasting state and at 2, 4, 6, and 8 h after an OFLT using human cytokine multiplex kits (Linco) and a Luminex LABScan™100 system. RESULTS: In the fasting state MIP-1ß, MIP-1α, IP-10, IFN-γ, MCP-1, and IL-8 were significantly increased in the FH group compared to the healthy controls (p <0.05). In addition, a significant decrease in postprandial chemokine plasma values were found in the FH group compared to fasting values after the OFLT. In normolipidemic nondiabetic controls no significant changes were found in the postprandial state. CONCLUSIONS: There was a postprandial decrease in chemokines related to early atherosclerotic processes after an OFLT in FH patients. These results confirm the influence of dietary patterns in this group of patients.
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Aterosclerosis/sangre , Aterosclerosis/complicaciones , Quimiocinas/sangre , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/complicaciones , Periodo Posprandial , Adolescente , Adulto , Anciano , Aterosclerosis/metabolismo , Índice de Masa Corporal , Estudios de Casos y Controles , Grasas Insaturadas en la Dieta/administración & dosificación , Grasas Insaturadas en la Dieta/farmacología , Ayuno/sangre , Femenino , Voluntarios Sanos , Humanos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/metabolismo , Masculino , Persona de Mediana Edad , Periodo Posprandial/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Despite the adverse effects of maternal smoking during pregnancy on the newborn's health are well-known, in the pediatric population, a high prevalence exists that is very much affected by second hand smoke (SHS). This study aims to investigate the impact of maternal smoking habits during pregnancy on cotinine levels in newborns during the first days of life. The high association between cotinine concentration in maternal and umbilical cord blood (UCB) has been previously reported, but the levels of blood cotinine that remain in infants born to smokers is unknown. METHODS: Cotinine concentration was measured in UCB, in maternal and newborn peripheral blood. Data from UCB sample dyads of ninety mothers and from seventy-one newborns were analyzed. RESULTS: Cotinine levels were significantly different among non-smokers (9.9 ± 5.9 ng/ml), moderate (67.3 ± 7.4 ng/ml), and heavy smokers (137.7 ± 19.5 ng/ml) (p<0.0001). Significant correlations were found between maternal and UCB cotinine (r=0.748; p<0.001), and between UCB and newborn plasma cotinine at 48 h after birth (r=0.541; p<0.001). The smokers exposed their infants to cotinine with a median of 31.7 ± 8.6 ng/ml (moderate) or 59.1 ± 13.3 ng/ml (heavy smokers) until at least, 48 h after birth. Reduced birth weight and length were significantly related with UCB cotinine levels. CONCLUSIONS: A positive association between UCB and plasmatic cotinine in newborns was found. The high cotinine levels detected in newborns from smoker mothers indicates that their infants are subjected to elevated SHS from birth. These results can help to reinforce the awareness of the adverse effects of smoking during pregnancy.
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Cotinina/sangre , Sangre Fetal/metabolismo , Intercambio Materno-Fetal/fisiología , Fumar/sangre , Contaminación por Humo de Tabaco/análisis , Adulto , Biomarcadores/sangre , Estudios de Cohortes , Femenino , Humanos , Recién Nacido , Embarazo , Fumar/efectos adversos , Factores de Tiempo , Adulto JovenRESUMEN
OBJECTIVES: Familial hypercholesterolemia (FH) is characterized by increased oxidative stress (OS) levels. In the postprandial state, lipids and lipoproteins modulate OS status through their impact on pro-oxidant and antioxidant mechanisms. The objective of this study was to evaluate in patients with FH the response to an unsaturated oral fat load test (OFLT) by analyzing the mRNA levels of genes involved in the glutathione and thioredoxin antioxidant systems. DESIGN AND METHODS: We analyzed 14 FH patients and 20 normolipidemic and normoglycemic controls. In both groups, mRNA values of antioxidant enzyme genes (glutathione and thioredoxin systems) were determined at baseline and at 2, 4, 6, and 8h after OFLT by real time PCR. RESULTS: In the fasting state the mRNA levels of antioxidant enzymes GPX4 and the GSR, GSS, and GCLC enzymes (involved in glutathione regeneration and synthesis) and thioredoxin (TXN), were significantly increased in the FH group compared to the healthy controls. Some genes (GPX1 and GPX4) were increased at 4h in both groups, but values for the rest of the antioxidant enzyme mRNAs were decreased in FH patients after 4h from unsaturated OFLT and were increased in controls. CONCLUSIONS: We concluded that an OFLT with predominantly unsaturated fat has a different effect on postprandial antioxidant enzyme mRNA levels in controls than in FH patients. Increased antioxidant enzyme mRNA is not the main way to reduce postprandial oxidative stress in FH. This difference could determine the influence of dietary patterns in these patients.
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Grasas Insaturadas en la Dieta/administración & dosificación , Glutatión/metabolismo , Hiperlipoproteinemia Tipo II/metabolismo , Estrés Oxidativo , Tiorredoxinas/metabolismo , Adulto , Ayuno , Femenino , Regulación Enzimológica de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Glutatión Peroxidasa/genética , Glutatión Reductasa/genética , Glutatión Sintasa/genética , Humanos , Hiperlipoproteinemia Tipo II/genética , Masculino , Persona de Mediana Edad , Oxidación-Reducción/efectos de los fármacos , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Autosomal dominant hypercholesterolemias (ADHs) are characterised by increased plasma levels of total and LDL cholesterol, predisposing to premature atherosclerosis. ADHs comprise several diseases with undistinguishable phenotype, caused by mutations in different genes: LDLR, APOB and PCSK9. Genetic studies are usually performed in patients with altered cholesterol levels. However, some persons carrying pathogenic mutations are normocholesterolemic and there are no further studies about this subject. We have studied the frequency of families and individuals carrying ADH mutations who do not present the disease in Spanish population. METHODS: We have analysed genes known to cause ADH by direct sequencing in 24 ADH families (215 members). Functional effect of some LDLR gene mutations was assessed by transfecting cultured cells with plasmids. RESULTS: Six families with mutations presented 7 mutation carriers who did not show ADH phenotype: 30% of ADH families presented normocholesterolemic individuals, and 7% of carriers of pathogenic mutations did not show ADH phenotype. We have analysed the effect of some of these mutations and they are responsible for impaired LDL receptor function. We have excluded mutations in APOB and PCSK9 genes that could reduce LDLc levels. CONCLUSIONS: An important percentage of ADH families presented individuals who do not show an ADH phenotype, but who are able to transmit the pathogenic mutation to their offspring. Genetic study of all subjects in ADH families should be performed in order to identify normocholesterolemic carriers that allow the detection of mutations in their descendants and the prevention of the disease consequences.
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Apolipoproteína B-100/genética , LDL-Colesterol/genética , Pruebas Genéticas/métodos , Hiperlipoproteinemia Tipo II/epidemiología , Hiperlipoproteinemia Tipo II/genética , Mutación , Adolescente , Adulto , Anciano , Animales , Apolipoproteínas B/genética , Células COS , Niño , Chlorocebus aethiops , Salud de la Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis , Linaje , Fenotipo , Proproteína Convertasa 9 , Proproteína Convertasas/genética , Análisis de Secuencia de ADN , Serina Endopeptidasas/genética , EspañaRESUMEN
Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.
Asunto(s)
Proliferación Celular , Glioblastoma/patología , Proteína de Retinoblastoma/metabolismo , Proteínas de Unión al GTP rho/fisiología , Citoesqueleto de Actina/química , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Ciclina D1/metabolismo , Citoesqueleto/química , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Transfección , Proteínas de Unión al GTP rho/genéticaRESUMEN
AP-1 (Activating Protein 1) transcription factor activity is tightly regulated at multiple levels, including dimer formation (i.e., Fos/Jun). Here we show that the intermediate filament protein lamin A/C suppresses AP-1 function through direct interaction with c-Fos, and that both proteins can interact and colocalize at the nuclear envelope (NE) in mammalian cells. Perinuclear localization of c-Fos is absent in Lmna-null cells but can be restored by lamin A overexpression. In vitro, preincubation of c-Fos with lamin A prior to the addition of c-Jun inhibits AP-1 DNA-binding activity. In vivo, overexpression of lamin A reduces the formation of c-Fos/c-Jun heterodimers, and suppresses AP-1 DNA-binding and transcriptional activity. Notably, c-Fos colocalizes with lamin A/C at the NE in starvation-synchronized quiescent cells lacking detectable AP-1 DNA binding. In contrast, serum-induced AP-1 DNA-binding activity coincides with abundant nucleoplasmic c-Fos expression without changes in lamin A/C localization. We also found that Lmna-null cells display enhanced proliferation. In contrast, lamin A overexpression causes growth arrest, and ectopic c-Fos partially overcomes lamin A/C-induced cell cycle alterations. We propose lamin A/C-mediated c-Fos sequestration at the NE as a novel mechanism of transcriptional and cell cycle control.