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ABSTRACT: We report a heteroplasmic novel mutation m.5658T>C in the mt-tRNA(Asn) gene in a patient who initially presented myopathy, bilateral ptosis and ophthalmoparesis and several years later developed a non-nephrotic proteinuria. The muscle biopsy showed cytochrome c oxidase (COX) negative and ragged red fibers and in the kidney biopsy that was taken in order to identify the causes of non-nephrotic proteinuria, a focal segmental glomerulosclerosis was observed. Using laser capture microdissection we isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that there was a clear increase in the mutation load in the COX negative muscle fibers. However, the low degree of mutation load found in the renal biopsy of the patient does not allow us to conclude that the m.5658T>C mutation is responsible for focal glomerulosclerosis. Additionally, we hypothesize that the mutated m.5658T nucleotide might be structurally relevant, as it is one of the fifteen nucleotides conserved in all the species analyzed and is situated contiguously to the discriminator base in the 3'end of the mt-tRNA, where the tRNase Z cleaves the 3' trailer sequence during mt-tRNA maturation.
Neuromuscular Disorders 01/2013; · 2.80 Impact Factor
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Nuria Garatachea, Tomàs Pinós,
Yolanda Cámara,
Gabriel Rodríguez-Romo,
Enzo Emanuele,
Giovanni Ricevuti,
Letizia Venturini,
Alejandro Santos-Lozano,
Catalina Santiago-Dorrego,
Carmen Fiuza-Luces,
Thomas Yvert,
Antoni L Andreu,
Alejandro Lucia
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ABSTRACT: The myostatin (MSTN) gene is a candidate to influence extreme longevity owing to its role in modulating muscle mass and sarcopenia and especially in inhibiting the main nutrient-sensing pathway involved in longevity, i.e. mammalian target of rapamycin. We compared allele/genotype distributions of the exonic MSTN variants K153R (rs1805086), E164K (rs35781413), I225T and P198A, in Spanish centenarians (cases, n = 156; 132 women, age range 100-111 years) and younger adults (controls, n = 384; 167 women, age <50 years). No subject of either group carried a mutant allele of the E164K, I225T or P198A variation. The frequency of the variant R allele was significantly higher in centenarians (7.1 %) than in controls (2.7 %) (P = 0.001). The odds ratio of being a centenarian if the subject had the R allele was 3.48 (95 % confidence interval 1.67-7.28, P = 0.001), compared to the control group, after adjusting for sex. The results were replicated in an Italian cohort (centenarians, n = 79 (40 women), age range 100-104 years; younger controls, n = 316 (155 women), age <50 years), where a higher frequency of the R allele in centenarians (7.6 %) compared to controls (3.0 %) (P = 0.004) was independently confirmed. Although more research is needed, the variant allele of the MSTN K153R polymorphism could be among the genetic contributors associated with exceptional longevity.
Age 01/2013; · 6.28 Impact Factor
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Gisela Nogales-Gadea, Tomàs Pinós,
Alejandro Lucia,
Joaquín Arenas,
Yolanda Camara,
Astrid Brull,
Noemí de Luna,
Miguel A Martín,
Elena Garcia-Arumí,
Ramon Martí,
Antoni L Andreu
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ABSTRACT: McArdle disease (glycogenosis type V), the most common muscle glycogenosis, is a recessive disorder caused by mutations in PYGM, the gene encoding myophosphorylase. Patients with McArdle disease typically experience exercise intolerance manifested as acute crises of early fatigue and contractures, sometimes with rhabdomyolysis and myoblobinuria, triggered by static muscle contractions or dynamic exercises. Currently, there are no therapies to restore myophosphorylase activity in patients. Although two spontaneous animal models for McArdle disease have been identified (cattle and sheep), they have rendered a limited amount of information on the pathophysiology of the disorder; therefore, there have been few opportunities for experimental research in the field. We have developed a knock-in mouse model by replacing the wild-type allele of Pygm with a modified allele carrying the common human mutation, p.R50X, which is the most frequent cause of McArdle disease. Histochemical, biochemical and molecular analyses of the phenotype, as well as exercise tests, were carried out in homozygotes, carriers and wild-type mice. p.R50X/p.R50X mice showed undetectable myophosphorylase protein and activity in skeletal muscle. Histochemical and biochemical analyses revealed massive muscle glycogen accumulation in homozygotes, in contrast to heterozygotes or wild-type mice, which did not show glycogen accumulation in this tissue. Additional characterization confirmed a McArdle disease-like phenotype in p.R50X/p.R50X mice, i.e. they had hyperCKaemia and very poor exercise performance, as assessed in the wire grip and treadmill tests (6% and 5% of the wild-type values, respectively). This model represents a powerful tool for in-depth studies of the pathophysiology of McArdle disease and other neuromuscular disorders, and for exploring new therapeutic approaches for genetic disorders caused by premature stop codon mutations.
Brain 07/2012; 135(Pt 7):2048-57. · 9.46 Impact Factor
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ABSTRACT: A human mitochondrial DNA (mtDNA) transition, m.1555A>G, in the 12S rRNA gene causes non-syndromic hearing loss. However, this pathological mutation is the wild-type allele in orangutan mtDNA. Here we rule out different genetic factors as the reason for its fixation in orangutans and show that aminoglycosides negatively affect the oxidative phosphorylation function by decreasing the synthesis of mtDNA-encoded proteins and the amount and activity of respiratory complex IV. These drugs also diminish the growth rate of orangutan cells. The m.1555G nucleotide is also the wild-type allele in other mammal species and they might be at risk of suffering a mitochondrial disorder if treated with aminoglycosides. Therefore, pharmacogenomic approaches should be used to confirm this possibility. These observations are important for human health. Due to the fact that old age and high frequency are criteria widely used in mitochondrial medicine to rule out a genetic change as being a pathological mutation, our results prevent against simplistic genetic approaches that do not consider the potential effect of environmental conditions. Hence, these results suggest that some ancient and highly frequent human population polymorphisms, such as those defining mtDNA haplogroups, in mitochondrial rRNA genes can be deleterious in association with new environmental conditions. Therefore, as the discovery of ribosomal antibiotics has allowed to fight infectious diseases and this breakthrough can be considered an important scientific advance or 'progress', our results suggest that 'progress' can also have a negative counterpart and render detrimental many of these mtDNA genotypes.
Human Molecular Genetics 08/2011; 20(21):4224-31. · 7.64 Impact Factor
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Gisela Nogales-Gadea, Tomàs Pinós,
Jonatan R Ruiz,
Pedro Femia Marzo,
Carmen Fiuza-Luces,
Ester López-Gallardo,
Eduardo Ruiz-Pesini,
Miguel Angel Martín,
Joaquín Arenas,
María Morán,
Antoni L Andreu,
Alejandro Lucia
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ABSTRACT: There is increasing evidence regarding the association between mitochondrial DNA (mtDNA) and aerobic capacity; however, whether mtDNA haplogroups are associated with the status of being an elite endurance athlete is more controversial. We compared the frequency distribution of mtDNA haplogroups among the following groups of Spanish (Caucasian) men: 102 elite endurance athletes (professional road cyclists, endurance runners), 51 elite power athletes (jumpers, throwers and sprinters), and 478 non-athletic controls. We observed a significant difference between endurance athletes and controls (Fisher exact test=17.89, P=0.015; Bonferroni's significant threshold=0.017), yet not between power athletes and controls (Fisher exact test=47.99, P=0.381) or between endurance and power athletes (Fisher exact test=5.53, P=0.597). We observed that the V haplogroup was overrepresented in endurance athletes (15.7%) compared with controls (7.5%) (odds ratio: 2.284; 95% confidence interval: 1.237, 4.322). In conclusion, our findings overall support the idea that mtDNA variations could be among the numerous contributors to the status of being an elite endurance athlete, whereas no association was found with elite power athletic status.
Mitochondrion 08/2011; 11(6):905-8. · 3.62 Impact Factor
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ABSTRACT: The cellular quality control systems enable surveillance and selective degradation of nonsense, nonstop, and no-go mRNAs. In the case of nonstop mRNA, different mechanisms of nonstop-mediated decay (NSD) have been described for bacteria, yeast and mammals, but the molecular consequences of nonstop mutations have been examined in only few cases of human disease. We describe a novel homozygous nonstop mRNA mutation (c.1416delC) in the TYMP gene encoding thymidine phosphorylase, in a patient with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In contrast to previous reports showing selective decay of pathogenic nonstop mRNAs, quantitative real-time PCR and 3'-RACE-RFLP analysis revealed unreduced nonstop mRNA levels in our patient and 2 heterozygous carriers of the mutation. The absence of thymidine phosphorylase protein in the homozygous patient, together with the partial decrease in levels of this protein in 2 carriers suggest that the main control system in this case resides at the translational or post-translational levels rather than through NSD. This is the first report showing an absence of NSD in a human disease, revealing that this surveillance mechanism has exceptions in vivo.
Human Mutation 04/2011; 32(4):E2061-8. · 5.69 Impact Factor
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Tomàs Pinós,
Gisela Nogales-Gadea,
Jonatan R Ruiz,
Gabriel Rodríguez-Romo,
Catalina Santiago-Dorrego,
Carmen Fiuza-Luces,
Félix Gómez-Gallego,
Amalia Cano-Nieto,
Nuria Garatachea,
María Morán,
Miguel Angel Martín,
Joaquín Arenas,
Antoni L Andreu,
Alejandro Lucia
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ABSTRACT: Mitochondrial haplogroups could influence individual susceptibility to mitochondrial DNA (mtDNA) damage, and human longevity, as indicated by previous studies with Caucasian (European) or Asian cohorts. Here, we compared the frequency of mtDNA haplogroups in a group of Spanish (Caucasian) centenarians (n = 65, aged 100-108 years, 58 women, most from the central part of Spain) and a group of healthy young adults (n = 138, 62 women, aged 20-40 years) of the same ethnic origin. We did not find significant differences between centenarians and the control group (P > 0.2). Only two centenarians (both women) had the haplogroup J, which hampered comparison with the control group (n = 15, five women). Our data confirm that the potential effects of mitochondrial haplogroups on human longevity might be population/geographic specific, with important differences between studies (notably, with regard to the previously reported potential benefit brought about by the haplogroup J) arising from the different living environment and ethnic background of the study cohorts.
Age 01/2011; 34(1):227-33. · 6.28 Impact Factor
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ABSTRACT: We report a heteroplasmic novel mutation m.5636T>C in the mt-tRNA(Ala) in a patient with bilateral ptosis and ophthalmoparesis in whom a muscle biopsy showed cytochrome c oxdidase (COX) negative and ragged red fibers. Using laser capture microdissection we have isolated COX negative fibers and COX positive fibers from the muscle of the patient and determined that the mutation load was clearly increased in COX negative muscle fibers. Additionally, the mutated m.5636T nucleotide is conserved in all the mammal and non-mammal species analyzed and might be structurally relevant as it is located in a position involved in the formation of tertiary structure of canonical mitochondrial tRNAs.
Mitochondrion 01/2011; 11(1):228-33. · 3.62 Impact Factor
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ABSTRACT: The human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5' UTR exons 1A and 1B differently regulate SHBG translation.
Cis-regulatory elements that could potentially regulate translation were identified within the 5'UTRs of SHBG TU-1A and TU-1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5'UTR sequence did show SHBG translation, whereas inclusion of the 5'UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5'UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B.
The use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.
PLoS ONE 01/2010; 5(11):e13844. · 4.09 Impact Factor
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ABSTRACT: Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.
Experimental Cell Research 06/2009; 315(17):3004-13. · 3.58 Impact Factor
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Antoni Hurtado, Tomàs Pinós,
Anna Barbosa-Desongles,
Sandra López-Avilés,
Jordi Barquinero,
Jordi Petriz,
Albert Santamaria-Martínez,
Joan Morote,
Inés de Torres,
Joaquim Bellmunt,
Jaume Reventós,
Francina Munell
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ABSTRACT: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERbeta) remain elusive.
We have analyzed the levels of ERbeta1 and ERbeta2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERbeta1 in the human prostate cancer LNCaP cell line.
Both ERbeta1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERbeta2 levels decreased during the S phase and increased in the G2/M phase. ERbeta1 protein was detected in both the nuclear and non-nuclear fractions, and ERbeta2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERbeta was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFkappaB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERbeta1 or ERbeta1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERbeta1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1-ERbeta1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested.
Our results demonstrate that, in LNCaP prostate cancer cells, both ERbeta isoforms are differentially expressed during the cell cycle and that ERbeta regulates the G1 phase by a non-genomic mechanism.
Cellular oncology: the official journal of the International Society for Cellular Oncology 02/2008; 30(4):349-65. · 4.17 Impact Factor
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ABSTRACT: Sex hormone-binding globulin (SHBG) binds steroids in the blood but is also present in the extravascular compartments of some tissues. Mice expressing a human SHBG transgene in the liver have human SHBG in their blood. In these animals, human SHBG accumulates within the stromal matrix of the endometrium and epididymis. This is remarkable because these tissues do not express the transgene. Human SHBG administered intravenously to wild-type mice in the presence of estradiol is rapidly sequestered within the endometrial stroma, and this prompted us to search for SHBG interacting proteins. Yeast two-hybrid screens revealed that fibulin-1D and fibulin-2 interact with the amino-terminal laminin G domain of SHBG. These interactions were verified in GST-pull down assays in which human SHBG bound the carboxyl-terminal domains of fibulin-1D and fibulin-2 in a steroid-dependent manner, with estradiol being the most effective ligand, and were enhanced by reducing the N-glycosylation of human SHBG. Like human SHBG, fibulin-1 and fibulin-2 concentrate within the endometrial stroma. In addition, SHBG co-immunoprecipitates with these fibulins in a proestrus uterine extract. These matrix-associated proteins may therefore sequester plasma SHBG within uterine stroma where it can control sex-steroid access to target cells. Given the interplay between fibulins and numerous proteins within the basal lamina, interactions between SHBG and matrix proteins may exert novel biological effects.
Journal of Biological Chemistry 07/2006; 281(23):15853-61. · 4.77 Impact Factor
