RESUMEN
The telomere length of human blastocysts exceeds that of oocytes and telomerase activity increases after zygotic activation, peaking at the blastocyst stage. Yet, it is unknown whether aneuploid human embryos at the blastocyst stage exhibit a different profile of telomere length, telomerase gene expression, and telomerase activity compared to euploid embryos. In present study, 154 cryopreserved human blastocysts, donated by consenting patients, were thawed and assayed for telomere length, telomerase gene expression, and telomerase activity using real-time PCR (qPCR) and immunofluorescence (IF) staining. Aneuploid blastocysts showed longer telomeres, higher telomerase reverse transcriptase (TERT) mRNA expression, and lower telomerase activity compared to euploid blastocysts. The TERT protein was found in all tested embryos via IF staining with anti-hTERT antibody, regardless of ploidy status. Moreover, telomere length or telomerase gene expression did not differ in aneuploid blastocysts between chromosomal gain or loss. Our data demonstrate that telomerase is activated and telomeres are maintained in all human blastocyst stage embryos. The robust telomerase gene expression and telomere maintenance, even in aneuploid human blastocysts, may explain why extended in vitro culture alone is insufficient to cull out aneuploid embryos during in vitro fertilization.
Asunto(s)
Telomerasa , Humanos , Telomerasa/genética , Telomerasa/metabolismo , Blastocisto/metabolismo , Embrión de Mamíferos/metabolismo , Aneuploidia , Telómero/genética , Telómero/metabolismoRESUMEN
PURPOSE: To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development. METHODS: Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p. RESULTS: LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P < 0.0001), suggesting that these 2-cell genes are activated at the early 2-cell stage. However, in early 2-cell embryos with AZT treatment, mRNA levels of Dux, Zscan4, and LINE-1 were significantly decreased. Furthermore, both Zscan4 and LINE-1 encoded proteins localized to telomere regions in 2-cell embryos, but this co-localization was dramatically reduced after AZT treatment (P < 0.001). CONCLUSIONS: Upon inhibition of LINE-1 retrotransposition in mouse 2-cell embryos, Dux, Zscan4, and LINE-1 were significantly downregulated, and telomere elongation was blocked. ZSCAN4 foci and their co-localization with telomeres were also significantly decreased, indicating that ZSCAN4 is an essential component of the telomere reprogramming that occurs in mice at the 2-cell stage. Our findings also suggest that LINE-1 may directly contribute to telomere reprogramming in addition to regulating gene expression.
Asunto(s)
Embrión de Mamíferos/fisiología , Desarrollo Embrionario/genética , Proteínas de Unión al ARN/genética , Telómero/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Desarrollo Embrionario/fisiología , Ratones , Células Madre Embrionarias de Ratones/fisiología , Cigoto/fisiologíaRESUMEN
BACKGROUND: Biochemical changes induced in red blood cells (RBCs) during storage may impair their function upon transfusion. Transfusion-associated stresses may further amplify storage lesion effects including increased phosphatidylserine (PS) exposure at the RBC membrane, microparticle (MP) release, and adhesion to endothelial cells (ECs). RBC stress susceptibility in vitro was investigated in relation to storage time and additive solution. STUDY DESIGN AND METHODS: Leukoreduced whole blood donations (n = 18) were paired, mixed, and resplit before separating the RBCs for storage in saline-adenine-glucose-mannitol (SAGM) or AS-1. Samples were taken after 3, 21, or 35 days. For oxidative stress treatment, RBCs were exposed to 0.5 mmol/L tert-butylhydroperoxide. Transfusion-associated stress was simulated by overnight culture at 37 °C with plasma containing inflammatory mediators. PS exposure and MPs were measured by flow cytometry and adhesion to ECs was tested under flow conditions. PS specificity of adhesion was tested by blocking with PS-containing lipid vesicles. RESULTS: Oxidative stress induced significantly higher PS exposure and adhesion to ECs in RBCs stored for 35 days compared to 3 days (p < 0.04). PS-containing vesicles blocked RBC-EC adhesion. After overnight culture with or without plasma, PS exposure and EC adhesion were significantly increased (p < 0.05). MP numbers increased with longer RBC storage and after RBC culture with plasma. Culture conditions influenced MP numbers from Day 35 RBCs. RBCs stored in SAGM had significantly higher PS exposure after stress treatment than AS-1 RBCs (p < 0.02). CONCLUSION: Storage for 35 days significantly increased RBC susceptibility to oxidative and in vitro transfusion-associated stresses and was higher for RBCs stored in SAGM compared to AS-1.
Asunto(s)
Adenina/farmacología , Conservación de la Sangre , Membrana Eritrocítica/metabolismo , Transfusión de Eritrocitos , Glucosa/farmacología , Manitol/farmacología , Estrés Oxidativo/efectos de los fármacos , Cloruro de Sodio/farmacología , Adhesión Celular/efectos de los fármacos , Técnicas de Cocultivo , Células Endoteliales/metabolismo , Femenino , Humanos , Masculino , Soluciones Farmacéuticas/farmacología , Fosfatidilserinas/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Microparticles (MPs) are small phospholipid-containing vesicles that have procoagulant properties. MPs are thought to contribute to the hemostatic potential of plasma. This study investigated the effects of whole blood (WB) hold time and leukoreduction (LR) on the MP profile and hemostatic potential of fresh-frozen plasma (FFP). STUDY DESIGN AND METHODS: WB units (n=12) from healthy donors were divided into two pairs and each pair was held at 20 to 24°C for 6 or 24 hours. At the designated hold time, 1 unit from the pair was LR while the other unit was not LR. FFP was prepared by standard procedures, aliquoted, and frozen. The MP content was determined by flow cytometry using an absolute count assay and specific labels for red blood cells (CD235a), platelets (CD41), and phosphatidylserine (PS). The hemostatic potential was determined by thrombelastography (TEG) and coagulation factor assays. RESULTS: Compared to non-LR-FFP, LR-FFP had significantly lower numbers of MPs, particularly CD41+ MPs and PS-positive MPs (p<0.03). LR-FFP, compared to non-LR-FFP, had a slower clot formation time (p=0.002); lower clot strength (p<0.001); and lower Factor (F)VIII, FXII, and fibrinogen levels (p<0.01). With longer WB hold time, the TEG profile was unchanged, although FVIII levels were decreased as expected (p<0.01). On average FFP units met quality requirements. CONCLUSION: LR of WB resulted in lower hemostatic potential of FFP in conjunction with depletion of MPs and coagulation factors. Longer WB hold time did not significantly affect the hemostatic potential of FFP as measured by TEG. Acceptable hemostatic quality was maintained for all FFP processing conditions studied.
Asunto(s)
Conservación de la Sangre/métodos , Micropartículas Derivadas de Células , Hemostasis , Procedimientos de Reducción del Leucocitos , Plasma/fisiología , Temperatura , Adulto , Recuento de Células Sanguíneas , Plaquetas/química , Plaquetas/fisiología , Factor VIII/análisis , Factor XIII/análisis , Fibrinógeno/análisis , Filtración , Citometría de Flujo , Humanos , Masculino , Lípidos de la Membrana/sangre , Persona de Mediana Edad , Fosfatidilserinas/sangre , Plasma/citología , TromboelastografíaRESUMEN
Expression of fetal γ-globin in adulthood ameliorates symptoms of ß-hemoglobinopathies by compensating for the mutant ß-globin. Reactivation of the silenced γ-globin gene is therefore of substantial clinical interest. To study the regulation of γ-globin expression, we created the GG mice, which carry an intact 183-kb human ß-globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ-globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta-gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human ß-globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase-1 in GG primary erythroid cells increases the proportion of eGFP(+) cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced >3-fold after treatment of erythroid precursors with epigenetic drugs known to induce γ-globin expression, demonstrating the suitability of the Gγ-globin eGFP reporter for evaluation of γ-globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the ß-globin locus and for discovery of novel therapies for the ß-hemoglobinopathies.
Asunto(s)
Eritropoyesis/fisiología , Regulación de la Expresión Génica , gamma-Globinas/metabolismo , Animales , Metilasas de Modificación del ADN/metabolismo , Modelos Animales de Enfermedad , Epigénesis Genética , Células Eritroides/metabolismo , Femenino , Hemoglobina Fetal/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Tiempo , Transgenes , Globinas beta/metabolismoRESUMEN
Microparticles (MPs) are shed from normal blood cells and may contribute to the coagulation potential of plasma. Transfusion of fresh frozen plasma (FFP) is used to correct coagulopathies and blood loss in trauma or major surgery. The role of MPs in FFP clinical efficacy is unknown. Regulations that govern the preparation of FFP vary in different countries. The aim of this study was to determine the effect of whole blood (WB)-hold conditions before FFP preparation on the MP profile. WB units were held at room temperature (RT) or combination of RT and refrigeration for up to 24h before FFP preparation. The MP content in thawed FFP was measured to reflect transfusion practice. The absolute number of MPs in FFP increased with longer WB hold time. Refrigeration of WB may also promote increased generation of MPs. In particular the number of platelet-derived and phosphatidylserine-containing MPs, which are known to have procoagulant properties, increased. Lipid peroxidation increased with longer WB-hold time. Donor-related factors appear to govern lipid peroxidation levels. Holistic proteomic and coagulant analyses of FFP MPs are warranted. Such information could guide the choice of the optimal handling conditions of WB and the most relevant quality control procedures for FFP. This article is part of a Special Issue entitled: Integrated omics.
Asunto(s)
Células Sanguíneas/metabolismo , Donantes de Sangre , Conservación de la Sangre , Micropartículas Derivadas de Células/metabolismo , Plasma/metabolismo , Adulto , Transfusión de Componentes Sanguíneos , Frío , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
A greater understanding of the regulatory mechanisms that govern γ-globin expression in humans, especially the switching from γ- to ß-globin, which occurs after birth, would help to identify new therapeutic targets for patients with ß-hemoglobinopathy. To further elucidate the mechanisms involved in γ-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human ß-globin locus under the control of (G)γ- or (A)γ-globin promoter and ß-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ-globin gene suppression.
Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , Globinas beta/genética , gamma-Globinas/genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Hemoglobina Fetal/genética , Colorantes Fluorescentes/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferencia de ARN , Proteínas Represoras , TransgenesRESUMEN
BACKGROUND: Ultraviolet radiation (UVR) is known to induce the activation of stress-inflammation signal transduction pathways, and to induce the activity of many proteases in skin cells. It is unknown whether the activation of proteases such as furin is related to changes in the phosphorylation status of p38MAPK. METHODS: The effect of UVR on immortalized keratinocyte (HaCaT) and squamous cell carcinoma (Colo16) cells was investigated with respect to cell survival, phosphorylation of p38MAPK, and the proprotein convertase, furin. The cells were exposed to either a low or a high dose of UVA and/or UVB and the viability was monitored over 48 h, along with changes in the intracellular expression of p38MAPK and furin. RESULTS: Low-dose UVA (2 kJ/m(2)) and/or UVB (0.2 kJ/m(2)) radiation had no effect on cell viability, except in UVA-irradiated Colo16 cells. High UVA (20 kJ/m(2)) caused a loss of cell viability in HaCaT cells, but not in Colo16 cells. The opposite effect was seen in cells exposed to a high UVB dose (2 kJ/m(2)). The viability of both cell cultures decreased when exposed to high-dose UVA+B radiation. UV irradiation downregulated the expression of phosphorylated p38 (pp38) in HaCaT cells irrespective of the UV dose and type. In Colo16 cells, UV radiation induced pp38 expression in the cells following exposure, with the highest increase in cells exposed to high-dose UVA. The expression of furin in UV-irradiated HaCaT cells was similar to that seen for pp38 expression. In Colo16 cells, UV radiation induced furin expression, with the highest increase seen in cells 24 h after exposure to both high-dose UVB and UVA+B radiation. CONCLUSION: The results show that there are differences between the effect of UV types and doses on cell function in the keratinocyte-derived cell lines examined in this study. The level of furin expression in Colo16 cells correlated to changes in pp38 levels in the cells following exposure to UV radiation, but not in HaCaT cells. From an improved understanding of the signalling pathways and their downstream events and how these may differ as a result of tumorigenesis, it may enable the development of inhibitors, which may have therapeutic applications.