RESUMEN
DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.
Asunto(s)
Análisis de los Alimentos , Industria de Procesamiento de Alimentos , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa/métodos , Alimentos Modificados Genéticamente , Plantas Modificadas Genéticamente/química , Glycine max/química , Glycine max/genética , Zea mays/química , Zea mays/genéticaRESUMEN
Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.
Asunto(s)
Canales Catiónicos TRPC/fisiología , Túnica Íntima/patología , Enfermedades Vasculares/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Hiperplasia , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Ratas , Ratas Endogámicas WKY , Vena Safena/patología , Porcinos , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Regulación hacia Arriba , Enfermedades Vasculares/tratamiento farmacológicoRESUMEN
Genetic programs controlling self-renewal and multipotentiality of stem cells have overlapping pathways with cell cycle regulation. Components of cell cycle machinery can play a key role in regulating stem cell self-renewal, proliferation, differentiation and aging. Among the negative regulators of cell cycle progression, the RB family members play a prominent role in controlling several aspects of stem cell biology. Stem cells contribute to tissue homeostasis and must have molecular mechanisms that prevent senescence and hold 'stemness'. RB can induce senescence-associated changes in gene expression and its activity is downregulated in stem cells to preserve self-renewal. Several reports evidenced that RB could play a role in lineage specification of several types of stem cells. RB has a role in myogenesis as well as in cardiogenesis. These effects are not only related to its role in suppressing E2F-responsive genes but also to its ability to modulate the activity of tissue-specific transcription factors. RB is also involved in adipogenesis through a strict control of lineage commitment and differentiation of adipocytes as well in determining the switch between brown and white adipocytes. Also, hematopoietic progenitor cells utilize the RB pathway to modulate cell commitment and differentiation. In this review, we will also discuss the role of the other two RB family members: Rb2/p130 and p107 showing that they have both specific and overlapping functions with RB gene.
Asunto(s)
Genes de Retinoblastoma , Células Madre/fisiología , Animales , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , División Celular , Senescencia Celular/fisiología , Embrión de Mamíferos/fisiología , Humanos , Mitosis , Proteína de Retinoblastoma/fisiología , Células Madre/citologíaRESUMEN
An increasing body of research is showing that cancers might contain their own stem cells. In fact, cancer cells, like stem cells, can proliferate indefinitely through a deregulated cellular self-renewal capacity. This raises the possibility that some features of tumor cells may be due to cancer stem cells. Stem cell-like cancer cells were isolated from several solid tumors. Now, evidence has shown that brain cancers, such as glioblastomas, medulloblastomas and astrocytomas, also contain cells that may be multipotent neural stem cell-like cells. In this review, we discuss the results of these studies, along with the molecular pathways that could be involved in cancer stem cell physiopathology.
Asunto(s)
Neoplasias Encefálicas/patología , Células Madre Neoplásicas/patología , Animales , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatología , Ciclo Celular , Separación Celular , Ensamble y Desensamble de Cromatina , Humanos , Modelos Biológicos , Células Madre Neoplásicas/fisiologíaRESUMEN
Five methodologies for extracting DNA from food samples are described. The food products analyzed are from either soybean or maize. They were selected on the basis of the mechanical, thermal, and chemical treatments that they had been subjected to during industrial processing. DNA preparations were evaluated for purity, yield, and average fragment size. Two endogenous genes, soybean lectin gene and alcohol dehydrogenase gene (adh1), were used to assess the degree of DNA degradation at different stages of the transformation chain. The goal of this study was to determine the role that extraction methods play in DNA amplification in order to select the best protocol for a food sample. This comparative evaluation can be specifically useful for detection of genetically modified ingredients in a variety of food matrices.
Asunto(s)
ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Análisis de los Alimentos/métodos , Glycine max/genética , Reacción en Cadena de la Polimerasa/métodos , Extracción en Fase Sólida/métodos , Zea mays/genética , Manejo de Especímenes/métodosRESUMEN
Marrow stromal stem cells (MSCs) are stem-like cells that are currently being tested for their potential use in cell therapy for a number of human diseases. MSCs can differentiate into both mesenchymal and nonmesenchymal lineages. In fact, in addition to bone, cartilage and fat, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes. RB and RB2/p130 genes are involved in the differentiation of several systems. For this reason, we evaluated the role of RB and RB2/p130 in the differentiation and apoptosis of MSCs under experimental conditions that allow for MSC differentiation toward the neuron-like phenotype. To this end, we ectopically expressed either RB or RB2/p130 and monitored proliferation, differentiation and apoptosis in rat primary MSC cultures induced to differentiate toward the neuron-like phenotype. Both RB and RB2/P130 decreased cell proliferation rate. In pRb-overexpressing cells, the arrest of cell growth was also observed in the presence of the HDAC-inhibitor TSA, suggesting that its antiproliferative activity does not rely upon the HDAC pathway, while the addition of TSA to pRb2/p130-overexpressing cells relieved growth inhibition. TUNEL reactions and studies on the expression of genes belonging to the Bcl-2 family showed that while RB protected differentiating MSCs from apoptosis, RB2/p130 induced an increase of apoptosis compared to controls. The effects of both RB and RB2/p130 on programmed cell death appeared to be HDAC- independent. Molecular analysis of neural differentiation markers and immunocytochemistry revealed that RB2/p130 contributes mainly to the induction of generic neural properties and RB triggers cholinergic differentiation. Moreover, the differentiation potentials of RB2/p130 and RB appear to rely, at least in part, on the activity of HDACs.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/fisiología , Neuronas/citología , Proteínas/fisiología , Proteína de Retinoblastoma/fisiología , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Adenoviridae/genética , Animales , Apoptosis/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/genética , Factores de Transcripción E2F , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Vectores Genéticos/genética , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/fisiología , Ácidos Hidroxámicos/farmacología , Inmunohistoquímica , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Ratas , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Factores de Transcripción/genética , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The complexity of the central nervous system (CNS) exposes it to a number of different diseases, often caused by only small variations in gene sequence or expression level. Antisense oligonucleotides and RNA interference-mediated therapies hold great promise for the treatment of CNS diseases in which neurodegeneration is linked to overproduction of endogenous protein or to synthesis of aberrant proteins coded by dominant mutant alleles. Nevertheless, difficulties related to the crossing of the blood-brain barrier, expression vectors, molecule design and to the choosing of the correct target, should be effectively solved. This review summarizes some of the most recent findings concerning the administration of potential nucleic acid-based therapeutic drugs, as well as the most promising studies performed both in vitro and in animal models of disease. Finally, some current clinical trials involving antisense oligonucleotides or silencing RNA for therapy of neurological disorders are illustrated. Results of current studies and clinical trials are exciting, and further results will be certainly reached with increasing knowledge of blood-brain barrier transporters, of genes involved in neurological disease and in new vectors for efficient delivery to brain.
Asunto(s)
Enfermedades del Sistema Nervioso/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Interferencia de ARN/fisiología , Animales , Ensayos Clínicos Fase II como Asunto , Modelos Animales de Enfermedad , Humanos , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/fisiopatologíaRESUMEN
We describe here a molecular method that can be used to detect genome traits of a given horticultural item at each stage from the farm to the market. We developed a procedure to extract and amplify by PCR DNA obtained from complex matrixes, such as dried figs and fig jam. Few fragmented DNA molecules can be recovered from food products. However, we were able to increase the yield of PCR reactions by successfully applying an enzymatic repair protocol to retrieved DNA.
Asunto(s)
ADN de Plantas/aislamiento & purificación , Ficus/genética , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Agar , Reacción en Cadena de la PolimerasaRESUMEN
Dot-blot and Northern-blot experiments, using strand-specific RNA probes, show that part of the bacteriophage T4 DNA that codes for six of the base plate structural genes (gp 51, 27, 28, 29, 48 and 54), is transcribed in vivo from both DNA strands. The r DNA strand transcripts contain sequences which are translated into structural proteins. Antisense l strand RNA is about 100 fold less abundant than RNA molecules transcribed from the r DNA strand.
Asunto(s)
Escherichia coli/genética , Genes Virales , Genes , Fagos T/genética , Transcripción Genética , Northern Blotting , ARN/genética , ARN sin Sentido , Mapeo Restrictivo , Proteínas Virales/genéticaRESUMEN
A reduction in mean erythrocyte volume has been reported in some strains of genetically hypertensive rat, and more recently it has been suggested that a similar alteration might be found in human essential hypertension. The relationship between erythrocyte volume and blood pressure was therefore studied in a random sample of an untreated male working population (n = 317; age 45.1 +/- 6.4 years, mean +/- s.d.). Neither systolic nor diastolic blood pressures were found to be related to erythrocyte volume (r = 0.022 and r = -0.014, respectively); in fact, erythrocyte volume was not different across quintiles of blood pressure. Smokers (n = 171) had lower blood pressure and a greater erythrocyte volume than non-smokers or ex-smokers (n = 144; 91.6 +/- 4.7 versus 88.2 +/- 5.5 fl; P less than 0.001), and heavy drinkers (greater than 110 g ethanol/day) had higher blood pressure and a greater erythrocyte volume compared with the rest of the study population (P less than 0.01). However, after adjustment of erythrocyte volume for these two potentially confounding factors, again no statistical association was found with blood pressure. The present study, therefore, does not support the hypothesis of a negative association between erythrocyte volume and blood pressure, whereas it confirms that the smoking habit and habitual alcohol intake are strong determinants of erythrocyte volume.
Asunto(s)
Presión Sanguínea , Volumen de Eritrocitos , Adulto , Consumo de Bebidas Alcohólicas , Análisis de Varianza , Antropometría , Estudios Transversales , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Fumar , Encuestas y CuestionariosRESUMEN
Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.
RESUMEN
Partial phosphorothioate (PS) antisense oligodeoxynucleotides (ODNs) targeted against rat AT1 receptor mRNA have been used to control blood pressure in normotensive (WKY) and spontaneously hypertensive (SHR) rats. Molecules were injected intracerebroventricularly (i.c.v., right lateral ventricle) in freely moving animals. The antisense ODN lowered the mean arterial pressure (MAP) 24 hours (-43 mmHg+/-10) and 48 hours (-30 mmHg+/-13) after injection, while the control ODN molecule had no significant effects. The observed decrease of blood pressure was due to a specific inhibition of AT1 receptor gene expression, since the level of its mRNA, monitored by reverse transcription (RT)- polymerase chain reaction (PCR), was significantly reduced by antisense molecule (-40%), compared to sense one. In normotensive rats no effect on MAP have been observed, while AT1 receptor gene expression is reduced (-40%) by antisense treatment. It is known that SHRs have an enhanced basal activity of the central renin-angiotensin system that induces an increase in central sympathetic outflow. Instead in WKY rats the central sympathetic outflow is not conditioned by the enhanced activity of brain renin-angiotensin system. Therefore in normotensive rats although partial PS ODN reduces the AT1 mRNA level this will not result in a modification of the sympathetic outflow and no change in MAP level would be observed.
Asunto(s)
Antagonistas de Receptores de Angiotensina , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Hipertensión/metabolismo , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMEN
OBJECTIVES: Remodelling and restenosis are complex biological processes responsible for bypass and percutaneous transluminal coronary angioplasty failures which are likely to affect many hundreds of genes. We evaluated the effectiveness of topically applied antisense oligonucleotides in reducing the translation of the messenger RNA for the transcription factor c-myc and in reducing stenosis. METHODS: Surgery was performed under sterile conditions; 60 Wistar-Kyoto male rats were anaesthetized by ketamine. The carotid arteries were isolated through a median incision in the anterior neck region. At the same point, 0.5 mm longitudinal incisions were performed. Haemostasis was obtained by an adventitial 8.0 stitch. Thirty animals were given 150 microg of c-myc antisense oligonucleotide (Group A) while the other 30 animals received 150 microg of c-myc control sense oligonucleotide (Group B). Oligo molecules were locally applied through 100 microl of 20% pluronic gel. Rats were sacrificed at 30 days; carotid arteries were explanted and stained. Qualitative histological analysis was performed in all cases; serial sections were made every 25 micro in seven consecutive rats for each group. Morphometric analysis was also performed, luminal and medial area values recorded and the ratio between the two areas calculated. Data from each animal were compared with the corresponding contralateral carotid artery and expressed as mean+/-standard deviation. Statistical comparison between the two groups was carried out by one-way ANOVA text. RESULTS: Qualitative histological analysis showed marked remodelling with complete disarray of vessel wall, neointima accumulation and evidence of elastic fibres in the adventitia of all animals of Group B versus Group A. Morphometric analysis showed a significant reduction in the lumen area in Group A animals together with increased values of the medial area versus Group B animals. In addition, the ratio between the lumen and medial area was significantly higher in Group A than in Group B (2.61+/-0.18 versus 1.14+/-0.33, P<0.0001). CONCLUSIONS: c-myc antisense oligonucleotides applied intraoperatively can reduce post-operative stenosis.
Asunto(s)
Arterias Carótidas/patología , Oligonucleótidos Antisentido/farmacología , Túnica Íntima/patología , Análisis de Varianza , Animales , Constricción Patológica/etiología , Constricción Patológica/prevención & control , Genes myc/genética , Genes myc/fisiología , Masculino , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian "Casti Amanti" house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4'-'6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.
Asunto(s)
Carpo Animal/química , ADN/análisis , Fémur/química , Histocitoquímica , Caballos/genética , Paleontología , Colorantes de Rosanilina , Animales , Carpo Animal/anatomía & histología , Cromomicina A3/química , Colorantes/química , Fémur/anatomía & histología , Colorantes Fluorescentes/química , Historia Antigua , Caballos/anatomía & histología , Humanos , Indoles/química , Sustancias Intercalantes/química , ItaliaRESUMEN
The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.
Asunto(s)
Bacteriófago T4/genética , Carboxipeptidasas/genética , Genes Virales , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón Iniciador/genética , Modelos Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN sin SentidoRESUMEN
Since the 1880s, a disparate and extensive literature has evolved examining the biologic effects of acidification on cells. More recently, effects on the health of human and other species of acidic agents contained, for example, in pollutants have been suggested, particularly relating to long-term exposures. This paper provides a review of the epidemiologic and toxicologic evidence concerning health effects--particularly carcinogenicity--attributable to sub-lethal acid exposure. Underlying biologic mechanisms that explain adverse health outcomes include pH modulation of toxicity for a number of xenobiotics (including carcinogens, genotoxins, and teratogens), and low-pH-induced changes of cells involving, for example, alterations in mitotic and enzyme regulation. More focused research is recommended to test the relationship between long-term exposures to acidic agents (with a consequent lowered cellular pH) and various health effects.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Enfermedades Profesionales/inducido químicamente , Ácidos Sulfúricos/toxicidad , Contaminantes Atmosféricos/análisis , Animales , Carcinógenos , Humanos , Concentración de Iones de Hidrógeno , Mitógenos , Ácidos Sulfúricos/análisis , TeratógenosRESUMEN
Neural stem cells (NSCs) raised the hope for cell-based therapies in human neurodevelopmental and neurodegenerative diseases. Current research strategies aim to isolate, enrich, and propagate homogeneous populations of neural stem cells. Unfortunately, several concerns with NSC cultures currently may limit their therapeutic promise. Exhaustion of growth factors and/or their uncontrolled release with burst and fall in their concentration may greatly affect the in vitro behavior of NSCs. In this context, we investigate whether a device containing heparan sulfate (HS), which is a co-factor in growth factor-mediated cell proliferation and differentiation, could potentiate and prolong the delivery of fibroblast growth factor-2 (FGF-2) and thus improve in vitro NSC cultivation. We demonstrated that NSCs cultivated in media with a controlled release of FGF-2 from a polyelectrolyte polymer showed a higher proliferation rate, and reduced apoptosis and senescence. In these experimental conditions NSCs preserve their stemness properties for a longer period of time compared with controls. Also of interest is that cell fate properties are conserved as well. The controlled release of FGF-2 reduced the level of oxidative stress and this is associated with a lower level of damaged DNA. This result may explain the reduced level of senescence and apoptosis in NSCs cultivated in the presence of hydrogel-releasing FGF-2.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células-Madre Neurales/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Electrólitos/química , Heparitina Sulfato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Cinética , Ratones , Células-Madre Neurales/citología , Polímeros/químicaRESUMEN
Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.
Asunto(s)
Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Cromatografía Liquida , Biología Computacional , Medios de Cultivo Condicionados/farmacología , Humanos , Inmunohistoquímica , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Espectrometría de Masas en TándemRESUMEN
Knowledge of the characteristics of the normal human aorta has been constrained by lack of data on fresh aortic tissue, especially from healthy individuals. In this study, the gene expression and morphological characteristics of the thoracic ascending aorta (AA) of healthy organ donors have been evaluated, with the aim of providing reference data for the analysis of pathological AAs. We analysed by RT-PCR the differential expression of mRNAs coding for myocardin, smoothelin, alpha-smooth muscle actin (alpha-SMA) and the ED-A isoform of fibronectin (ED-A FN) in AA specimens from donors, integrating the results with immunohistochemical analysis of the same targets. Morphological and morphometric characteristics of the AAs were also evaluated. In order to account for possible regional variations in wall structure, the convexity of the aortic profile was compared to the concavity. No differences in gene expression occurred for any of the target genes between the concavity and the convexity of AAs. Immunohistochemistry revealed a different distribution of total FN and of its ED-A isoform in the media and in the intima. Smoothelin is expressed by the majority of cells in the media, with some positive cells also in the intima. Alpha-SMA is expressed in all the tunicae. Immunohistochemistry also revealed in the convexity of 50% of AAs the presence of discrete areas in the subadventital media with altered structure and cell morphology and with altered gene expression, resulting positive for ED-A FN and alpha-SMA, but not for smoothelin, indicating the occurrence of early lesions also in macroscopically healthy AAs.
Asunto(s)
Aorta Torácica/anatomía & histología , Aorta Torácica/química , Actinas/análisis , Actinas/genética , Adulto , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/genética , Femenino , Fibronectinas/análisis , Fibronectinas/genética , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas Musculares/análisis , Proteínas Musculares/genética , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/análisis , Transactivadores/genética , Adulto JovenRESUMEN
We focused our attention on brahma-related gene 1 (BRG1), the ATPase subunit of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, and analyzed its role in mesenchymal stem cell (MSC) biology. We hypothesized that deviation from the correct concentration of these proteins, which act at the highest level of gene regulation, may be deleterious for cells. We wanted to know what would happen if a cell had to cope with altered regulation of gene expression, either by upregulation or downregulation of BRG1. We assumed that cells would try to restore homeostasis or, alternatively, that the event could trigger senescence/apoptosis phenomena. To this end, in MSCs, we silenced BRG1gene. Knockdown of BRG1 expression induced a significant increase in senescent cells and decrease in apoptotic cells. It is interesting that BRG1 downregulation also induced an increase in heterochromatin. At the molecular level, these phenomena were associated with activation of retinoblastoma-like protein 2 (RB2)/P130- and P53-related pathways. Senescence was accompanied by reduced expression of some stemness-related genes. This is consistent with our previous research, which showed that BRG1 upregulation by ectopic expression also induced senescence processes. Together, these data suggest that BRG1 belongs to a class of genes whose expression is tightly regulated; hence, subtle alterations in BRG1 activity seem to negatively affect mechanisms regulating chromatin status and, in turn, impair cellular physiology.