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Telomerase reverse transcriptase (TERT) (catalytic subunit of telomerase) is linked to the development of coronary artery disease (CAD); however, whether the role of nuclear vs. mitchondrial actions of TERT is involved is not determined. Dominant-negative TERT splice variants contribute to decreased mitochondrial integrity and promote elevated reactive oxygen species production. We hypothesize that a decrease in mitochondrial TERT would increase mtDNA damage, promoting a pro-oxidative redox environment. The goal of this study is to define whether mitochondrial TERT is sufficient to maintain nitric oxide as the underlying mechanism of flow-mediated dilation by preserving mtDNA integrity.Immunoblots and quantitative polymerase chain reaction were used to show elevated levels of splice variants α- and ß-deletion TERT tissue from subjects with and without CAD. Genetic, pharmacological, and molecular tools were used to manipulate TERT localization. Isolated vessel preparations and fluorescence-based quantification of mtH2O2 and NO showed that reduction of TERT in the nucleus increased flow induced NO and decreased mtH2O2 levels, while prevention of mitochondrial import of TERT augmented pathological effects. Further elevated mtDNA damage was observed in tissue from subjects with CAD and initiation of mtDNA repair mechanisms was sufficient to restore NO-mediated dilation in vessels from patients with CAD. The work presented is the first evidence that catalytically active mitochondrial TERT, independent of its nuclear functions, plays a critical physiological role in preserving NO-mediated vasodilation and the balance of mitochondrial to nuclear TERT is fundamentally altered in states of human disease that are driven by increased expression of dominant negative splice variants.
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Enfermedad de la Arteria Coronaria , Telomerasa , Humanos , Telomerasa/genética , Peróxido de Hidrógeno/metabolismo , Enfermedad de la Arteria Coronaria/genética , Vasodilatación , Oxidación-ReducciónRESUMEN
Bone mechanobiology is the study of the physical, biological and mechanical processes that continuously affect the multiscale multicellular system of the bone from the organ to the molecular scale. Current knowledge derives from experimental studies, which are often limited to gathering qualitative data in a cross-sectional manner, up to a restricted number of time points. Moreover, the simultaneous collection of information about 3D bone microarchitecture, cell activity as well as protein distribution and level is still a challenge. In silico models can expand qualitative information with hypothetical quantitative systems, which allow quantification, testing and comparison to existing quantifiable experimental data. An overview of multiscale, multiphysics, agent-based and hybrid techniques and their applications to bone mechanobiology is provided in the present review. The study analysed how mechanical signals, cells and proteins can be modelled in silico to represent bone remodelling and adaptation. Hybrid modelling of bone mechanobiology could combine the methods used in multiscale, multiphysics and agent-based models into a single model, leading to a unified and comprehensive understanding of bone mechanobiology. Numerical simulations of in vivo multicellular systems aided in hypothesis testing of such in silico models. Recently, in silico trials have been used to illustrate the mechanobiology of cells and signalling pathways in clinical biopsies and animal bones, including the effects of drugs on single cells and signalling pathways up to the organ level. This improved understanding may lead to the identification of novel therapies for degenerative diseases such as osteoporosis.
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Huesos , Modelos Biológicos , Animales , Biofisica/métodos , Simulación por Computador , Estudios TransversalesRESUMEN
Aims Burkitt Lymphoma (BL) accounts for approximately 40% of childhood non-Hodgkin Lymphoma (NHL) in the developed world. Survival rates have improved dramatically in recent years, a success attributed to better use of poly-chemotherapy and targeted immunotherapy. Nevertheless, relapse is unpredictable and carries a dismal prognosis. We report on event-free survival (EFS) and overall survival (OS) rates in the Republic of Ireland (ROI) during 2000-2017, and evaluate novel predictors of outcome. Methods Data was collected by retrospective review of patient medical records. Results Thirty-three patients were identified (twenty-five [76%] males, eight [24%] females), fourteen [42%] having stage III disease at presentation. Six [18%] had stage IV disease. Five [15%] had refractory disease; one salvaged with allogeneic stem cell transplantation. Of the four [12%] who died; two [50%] had weights >99th centile, one [25%] >90th centile. One died during induction from refractory lactic acidosis, one from early relapse. Discussion EFS and OS was 85% and 89% respectively; in keeping with the best international standards. Obesity appears to be a poor predictor of outcome in our cohort.
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Linfoma de Burkitt , Adolescente , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/epidemiología , Linfoma de Burkitt/terapia , Niño , Estudios de Cohortes , Femenino , Humanos , Inmunoterapia , Masculino , Obesidad , Estudios RetrospectivosRESUMEN
Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.
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Células Madre Embrionarias/ultraestructura , Células Madre Pluripotentes Inducidas/ultraestructura , Mitocondrias/ultraestructura , Membranas Mitocondriales/ultraestructura , Animales , Microscopía Electrónica de Transmisión/métodosRESUMEN
Pluripotent stem cells (PSCs) have been described in naïve or primed pluripotent states. Domestic dogs are useful translational models in regenerative medicine, but their embryonic stem cells (cESCs) remain narrowly investigated. Primed-like cESCs expanded in the presence of leukemia inhibitory factor and fibroblast growth factor 2 (LIF-FGF2) acquire features of naïve pluripotency when exposed to chemical inhibitors and LIF (2iL). However, proliferation of cESCs is influenced by the pluripotent state and is comparatively slower than human or mouse PSCs. We propose that different metabolic pathway activities support ATP generation and biomass accumulation necessary for LIF-FGF2 and 2iL cESC proliferation. We found that 2iL cESCs have greater respiratory capacity, altered mitochondrial chain complex stoichiometry and elevated mitochondrial polarization state. Yet, 2iL-enriched cESCs exhibited immature ultrastructure, including previously unrecognized changes to cristae organization. Enhanced ATP level in 2iL cESCs is associated with altered retrograde signalling, whereas LIF-FGF2 cESCs exhibit a lipogenic phenotype. Inhibition of oxidative phosphorylation impaired proliferation and ATP production in 2iL cESCs but not LIF-FGF2 cESCs, which remained sensitive to glycolysis inhibition. Our study reveals distinct bioenergetic mechanisms contributing to steady-state expansion of distinct canine pluripotent states that can be exploited to improve derivation and culture of canine PSCs.
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Células Madre Embrionarias/metabolismo , Mitocondrias/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Perros , Células Madre Embrionarias/citología , Humanos , Fosforilación Oxidativa , Células Madre Pluripotentes/citologíaRESUMEN
Background: Chromosomal trisomies are associated with advancing maternal age. In Ireland, information on the total prevalence and outcome of trisomy affected pregnancies is unavailable. This study aimed to ascertain more precise data on Trisomies 21, 18 and 13 in a large Irish region during the period 2011-2013. Methods: Multiple information sources were used in case finding, including a regional congenital anomaly register, all maternity and paediatric hospitals in the region and the regional Department of Clinical Genetics. Results: There were 394 trisomy cases from 80 894 total births, of which 289 were Trisomy 21, 75 were Trisomy 18 and 30 were Trisomy 13. The total prevalence rate was 48.9/10 000 births, 35.7, 9.3 and 3.7 for Trisomies 21, 18 and 13, respectively. Over 90% of Trisomies 18/13 and 47% of Trisomy 21 were diagnosed prenatally; 61% of Trisomy 21 cases and nearly 30% of Trisomies 18/13 were live births; 38% all trisomy affected pregnancies ended in a termination. Conclusions: This study provides precise data on the total prevalence and outcome of trisomy affected pregnancies in the East of Ireland. Total prevalence rates were higher than previously reported. Prenatal diagnosis had a significant impact on outcome. These data provide a better basis for planning of services for live-born children affected by trisomy.
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Trastornos de los Cromosomas/epidemiología , Trisomía , Adulto , Trastornos de los Cromosomas/diagnóstico , Trastornos de los Cromosomas/genética , Síndrome de Down/diagnóstico , Síndrome de Down/epidemiología , Femenino , Humanos , Irlanda/epidemiología , Edad Materna , Embarazo , Resultado del Embarazo/epidemiología , Diagnóstico Prenatal/estadística & datos numéricos , Prevalencia , Trisomía/genética , Síndrome de la Trisomía 13/diagnóstico , Síndrome de la Trisomía 13/epidemiología , Síndrome de la Trisomía 18/diagnóstico , Síndrome de la Trisomía 18/epidemiología , Adulto JovenRESUMEN
BACKGROUND: Investigation of patients, particularly children, with unexplained global developmental delay (GDD)/learning disability (LD) has been challenging due to a lack of clear guidance from specialised centres. Limited knowledge of rare diseases and a poor understanding of the purpose or limitations of appropriate investigations have been some of the principal reasons for this difficulty. AIMS: A guideline development group was formed to recommend on appropriate, first line metabolic, genetic and radiological investigations for children and adults with unexplained GDD/ID. METHODS AND RECOMMENDATIONS: A comprehensive literature search was conducted, evaluated and reviewed by the guideline committee and a best practice protocol for first line assessment and genetic, metabolic and radiological investigations was decided upon after considering diagnostic yield, practicality, treatability and costs. CONCLUSION: It is hoped that these recommendations will become national guidelines for the first line metabolic, genetic and radiological investigation of patients presenting with unexplained GDD/ID.
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Discapacidades del Desarrollo/diagnóstico , Discapacidades para el Aprendizaje/diagnóstico , Errores Innatos del Metabolismo/diagnóstico , Adulto , Niño , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Humanos , Discapacidades para el Aprendizaje/genética , Discapacidades para el Aprendizaje/metabolismo , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/metabolismo , Enfermedades RarasRESUMEN
OBJECTIVES: To examine what experienced acupuncture practitioners and researchers considered key aspects of treatment to promote cephalic version for women with a breech presentation, and to establish a treatment protocol through consensus to guide the self administration of moxa by pregnant women. METHODS AND DESIGN: The Delphi method was used to seek the opinions of key informants. Sixteen English speaking international, Australian and New Zealand acupuncturists working in the area of pregnancy were invited to participate in the study. Participants were given a link to an online survey, and their views sought on treatment parameters guiding the treatment of breech presentation within a research setting. RESULTS: Two rounds of the Delphi process were undertaken, 12 participants completed round one, and 10 completed round two. Eighty percent of participants agreed that moxa should commence between 34 and 35 weeks gestation. Ninety percent agreed to self administration of moxa by the woman, and use of smokeless and odourless sticks. Seventy percent agreed moxa should be applied for a minimum of 10 days, and be applied once a day for 30min. Monitoring safety was identified as an important outcome. Ninety percent agreed study clinical outcomes should assess side effects including burns, and maternal and foetal outcomes. CONCLUSION: Findings from our study promote the clinical validity for a future research protocol, and highlight other areas for research to evaluate the role of acupuncture and moxibustion with normalising birth.
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Presentación de Nalgas/terapia , Moxibustión/métodos , Versión Fetal/métodos , Terapia por Acupuntura , Femenino , Personal de Salud/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Embarazo , Proyectos de InvestigaciónRESUMEN
Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of pre-implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed.
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Técnicas de Cultivo de Embriones/veterinaria , Embrión de Mamíferos/fisiología , Células Madre Pluripotentes/fisiología , Técnicas Reproductivas Asistidas/veterinaria , Animales , Desarrollo EmbrionarioRESUMEN
We report a case of Albright hereditary osteodystrophy (AHO) in a three-year-old girl with a microduplication at 17q11.2. The child developed obesity within the first 6 months of life. A diagnosis of Albright was made at age 2 years when biochemical evidence of parathyroid resistance was found. No mutations were identified in guanine nucleotide-binding protein G (s) subunit alpha (GNAS1). Subsequent investigations revealed methylation disturbance at GNAS1A, neuroendocrine secretory protein antisense (NESPAS) and neuroendocrine secretory protein 55 (NESP55) confirming a diagnosis of pseudohypothyroidism type 1B. A deletion of NESP55 and uniparental disomy chromosome 20 were excluded which suggested that the features of AHO arose through a purely epigenetic mechanism. Further investigation revealed a de novo microduplication at 17q11.2 encompassing the neurofibromatosis type 1 (NF1) gene. The combination of two rare de novo events in the same child raises the possibility that duplication of a gene within the 17q11.2 region may have triggered abnormal methylation in the GNAS cluster region on chromosome 20.
RESUMEN
The present study compared developmental potential, telomerase activity and transcript levels of X-linked genes (HPRT, MECP2, RPS4X, SLC25A6, XIAP, XIST and ZFX) in bovine somatic cell nuclear transfer (SCNT) embryos reconstructed with cells derived from a freemartin (female with a male co-twin) or from normal female cattle (control). The rates of cleavage, development to blastocyst and hatched blastocyst stage, and the mean numbers of total and inner cell mass cells in the freemartin SCNT embryos were not significantly different from those of control SCNT embryos (p > 0.05). The levels of telomerase activity analyzed by RQ-TRAP in the freemartin SCNT embryos were also similar to those of the normal SCNT embryos. Transcript levels of HPRT, MECP2, RPS4X and XIAP, measured by quantitative real-time RT-PCR, were not significantly different between the control and freemartin SCNT embryos (p > 0.05). However, the transcript levels of SLC25A6, XIST and ZFX were significantly decreased in the freemartin SCNT embryos compared to control SCNT embryos (p < 0.05). Transfer of 71 freemartin SCNT embryos to 22 recipient cows resulted in 4 (18%) pregnancies, which were lost between days 28 and 90 of gestation. Taken together, the present study demonstrates that the transcript levels of several X-linked genes, especially XIST, showed an aberrant pattern in the freemartin SCNT embryos, suggesting aberrant X inactivation in freemartin clones which may affect embryo survival.
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Embrión de Mamíferos/metabolismo , Freemartinismo/genética , Genes Ligados a X/genética , Técnicas de Transferencia Nuclear/veterinaria , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Bovinos , Clonación de Organismos , Transferencia de Embrión/veterinaria , Desarrollo Embrionario , Femenino , Muerte Fetal/genética , Muerte Fetal/veterinaria , Masculino , Embarazo , ARN Largo no Codificante , ARN Mensajero/análisis , ARN no Traducido/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Telomerasa/metabolismoRESUMEN
OBJECTIVE: To investigate the osteogenic differentiation potential of equine umbilical cord blood-derived multipotent mesenchymal stromal cells (CB-MSC) within coralline hydroxyapatite scaffolds cultured in osteogenic induction culture medium. METHODS: Scaffolds seeded with equine CB-MSC were cultured in cell expansion culture medium (control) or osteogenic induction medium (treatment). Cell viability and distribution were confirmed by the MTT cell viability assay and DAPI nuclear fluorescence staining, respectively. Osteogenic differentiation was evaluated after 10 days using reverse transcription polymerase chain reaction, alkaline phosphatase activity, and secreted osteocalcin concentration. Cell morphology and matrix deposition were assessed by scanning electron microscopy (SEM) after 14 days in culture. RESULTS: Cells showed viability and adequate distribution within the scaffold. Successful osteogenic differentiation within the scaffolds was demonstrated by the increased expression of osteogenic markers such as Runx2, osteopontin, osteonectin, collagen IA; increased levels of alkaline phosphatase activity; increased osteocalcin protein secretion and bone-like matrix presence in the scaffold pores upon SEM evaluation. CLINICAL SIGNIFICANCE: These results demonstrate that equine CB-MSC maintain viability and exhibit osteogenic potential in coralline hydroxyapatite scaffolds when induced in vitro . Equine CB-MSC scaffold constructs deserve further investigation for their potential role as biologically active fillers to enhance bone-gap repair in the horse.
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Diferenciación Celular , Cerámica/química , Sangre Fetal/citología , Hidroxiapatitas/química , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Osteogénesis/fisiología , Animales , Supervivencia Celular , ADN/biosíntesis , Ensayo de Inmunoadsorción Enzimática/veterinaria , Regulación de la Expresión Génica , Caballos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Microscopía Electrónica de Rastreo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Osteocalcina/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Ingeniería de Tejidos/métodosRESUMEN
Enucleation of a recipient oocyte is an important essential process in the procedure of somatic cell nuclear transfer (SCNT). The present study investigated a method for the improvement of enucleation efficiency. Oocytes were denuded of cumulus cells before the completion of nuclear maturation (pre-denuded) after 12 h of culture at MI stage and subsequently cultured for additional 6 h until the completion of nuclear maturation and extrusion of the first polar body (PB1). The extrusion rate of PB1 was not significantly different in the pre-denuded oocyte group, compared with control oocyte group matured for 18 h. However, the number of oocytes showing the metaphase II (MII) located just underneath the PB1 was significantly higher (p<0.05) in the pre-denuded oocyte group than those in control oocyte group. To test the effect of pre-denuding on the enucleation rate and developmental potential of embryos to blastocyst stage, subsequent somatic cell nuclear transfer comparisons were made with three different methods of enucleation at MII stage using vital dyes (demicoline and Hoescht) or the PB1 (blind enucleation) to localize the chromosome plate. Enucleation rate of the oocytes with demicoline, Hoechst and pre-denuding enucleation groups were significantly higher (p<0.05) than those of blind enucleation groups. However, cleavage rate to two-cell stage and, developmental rate to blastocyst and hatched blastocyst stage, the mean numbers of total and ICM cells in the SCNT embryos with Hoechst enucleation groups were significantly decreased (p<0.05), compared to those of blind, demicoline and pre-denuding enucleation groups. Moreover, the level of telomerase activity was also significantly (p<0.05) decreased in SCNT blastocysts of Hoechst enucleation group, compared to those of blind, demicoline and pre-denuding enucleation groups. Taken together, pre-denuding enucleation group using pre-denuded oocytes was a useful and simple enucleation method for bovine SCNT embryos.
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Bovinos/embriología , Clonación de Organismos/veterinaria , Técnicas de Cultivo de Embriones/veterinaria , Técnicas de Transferencia Nuclear/veterinaria , Oocitos/citología , Animales , Núcleo Celular , Fibroblastos , Cuerpos PolaresRESUMEN
OBJECTIVE: Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenchymal stromal cells (MSC) have been derived from multiple sources in the horse including bone marrow and umbilical cord blood. The objective of this study was to provide an in vitro comparison of the chondrogenic potential in MSC derived from adult bone marrow (BM-MSC) and umbilical cord blood (CB-MSC). RESULTS: MSC from both sources produced tissue with cartilage-like morphology that stained positive for proteoglycans and expressed cartilage markers. The CB-MSC pellets were larger and showed hyaline-like cartilage morphology as early as day six. Gene expression of collagen type 21, aggrecan and CD-RAP was higher in CB- than BM-MSC pellets. Expression of Sox9 mRNA was similar between CB- and BM-MSC pellets. Protein concentration of cartilage-derived retinoic acid sensitive protein was higher in culture medium from CB- than BM-MSC pellets. CONCLUSION: CB-MSC and BM-MSC were both capable of producing hyaline-like cartilage in vitro . However, in this study the MSC from umbilical cord blood appeared to have more chondrogenic potential than the BM-MSC based on the cells tested and parameters measured.
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Células de la Médula Ósea/fisiología , Condrogénesis/fisiología , Sangre Fetal/citología , Caballos/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Animales , Células Cultivadas , Criopreservación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiologíaRESUMEN
Developmental arrest is one of the mechanisms responsible for the elevated levels of embryo demise during the first week of in vitro development. Approximately 10-15% of IVF embryos permanently arrest in mitosis at the 2- to 4-cell cleavage stage showing no indication of apoptosis. Reactive oxygen species (ROS) are implicated in this process and must be controlled in order to optimize embryo production. A stress sensor that can provide a key understanding of permanent cell cycle arrest and link ROS with cellular signaling pathway(s) is p66Shc, an adaptor protein for apoptotic-response to oxidative stress. Deletion of the p66Shc gene in mice results in extended lifespan, which is linked to their enhanced resistance to oxidative stress and reduced levels of apoptosis. p66Shc has been shown to generate mitochondrial H(2)O(2) to trigger apoptosis, but may also serve as an integration point for many signaling pathways that affect mitochondrial function. We have detected elevated levels of p66Shc and ROS within arrested embryos and believe that p66Shc plays a central role in regulating permanent embryo arrest. In this paper, we review the cellular and molecular aspects of permanent embryo arrest and speculate on the mechanism(s) and etiology of this method of embryo demise.
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Embrión de Mamíferos/metabolismo , Proteínas Adaptadoras de la Señalización Shc/fisiología , Transducción de Señal/fisiología , Animales , Apoptosis/genética , Apoptosis/fisiología , Embrión de Mamíferos/citología , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica , Humanos , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal/genéticaRESUMEN
Methylation of specific CG residues in the mammalian genome results in tissue-specific patterns of gene expression, which are critical for cell differentiation. Embryos that fail to establish and maintain proper DNA methylation patterns show severe developmental abnormalities as is the case of DNA methyltransferase 1 (Dnmt1) -deficient embryos. Dnmt1 is the main maintenance methyltransferase in the mouse and its expression is regulated by a splicing mechanism that dictates the expression of stage-specific isoforms. Little is known about Dnmt1 expression in the cow and isoforms of Dnmt1 are yet unknown in this species. Here we demonstrate that the previously described bovine Dnmt1 transcript is ubiquitously expressed in embryos and fetal tissue. In addition, we report the identification of a splice variant of the bovine Dnmt1, which shows a ubiquitous expression pattern. This new transcript was detected using 5'RACE and genomic mapping and its expression pattern was shown to be consistent with a tissue-specific mode of regulation. Furthermore, our analysis shows that the expression of an oocyte-specific isoform of Dnmt1 is unlikely to occur in cattle. The newly reported isoform of Dnmt1 was demonstrated to be, similarly to Dnmt1a, polyadenylated and if translated possess the functional domains necessary for maintenance and de novo methyltransferase activity.
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Empalme Alternativo , ADN (Citosina-5-)-Metiltransferasas , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Isoformas de Proteínas , Animales , Secuencia de Bases , Bovinos , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribución TisularRESUMEN
Early pregnancy diagnosis and monitoring play an important role following embryo transfer in sheep. The aims of the current study were to investigate (i) the pattern of serum progesterone profiles in sheep carrying somatic cell nuclear transfer (SCNT)-derived (clone) pregnancies, and (ii) the frequency of pregnancy loss during development following SCNT embryo transfer. Sheep SCNT embryos were made using standard nuclear transfer techniques. Day 7 embryos were surgically transferred to oestrus-synchronized recipients (n = 27). As a control, normal fertile ewes (n = 12) were bred by natural breeding. Serum was collected from all the ewes on the day of estrus (day 0 sample), 7 days post-estrus (day 7 sample) and 19 days post-estrus (day 19 sample) and every 10 days thereafter until lambing or pregnancy loss occurred. Serum progesterone (P4) was assessed using enzyme immunoassay. Pregnancy was confirmed by ultrasound scanning on day 35 of pregnancy followed by subsequent scanning every 10 days. In control ewes, pregnancy rate on day 35 was 83.3% (10/12), whereas in the ewes that received SCNT embryos, it was 22.2% (6/27; p < 0.05). The day 45 pregnancy rate in the control ewes was 83.3%, whereas in the SCNT embryo recipients it was 11.0% (p < 0.05). Hormone analysis revealed that SCNT embryo recipients exhibited a significantly lower P4 profiles at different time points in pregnancy compared to controls (p < 0.05). This study highlights the use of serum progesterone in combination with ultrasound for the investigation of embryo loss and crucial times during development of normal and SCNT embryos in sheep. Further, the serum P4 levels directly reflect the degree of placental development in these two groups.