RESUMEN
Cancer cells reprogram their metabolism to maintain both viability and uncontrolled proliferation. Although an interplay between the genetic, epigenetic and metabolic rewiring in cancer is beginning to emerge, it remains unclear how this metabolic plasticity occurs. Here, we report that in prostate cancer cells (PCCs) microRNAs (miRNAs) greatly contribute to deregulation of mitochondrial fatty acid (FA) oxidation via carnitine system modulation. We provide evidence that the downregulation of hsa-miR-124-3p, hsa-miR-129-5p and hsa-miR-378 induced an increase in both expression and activity of CPT1A, CACT and CrAT in malignant prostate cells. Moreover, the analysis of human prostate cancer and prostate control specimens confirmed the aberrant expression of miR-124-3p, miR-129-5p and miR-378 in primary tumors. Forced expression of the miRNAs mentioned above affected tumorigenic properties, such as proliferation, migration and invasion, in PC3 and LNCaP cells regardless of their hormone sensitivity. CPT1A, CACT and CrAT overexpression allow PCCs to be more prone on FA utilization than normal prostate cells, also in the presence of high pyruvate concentration. Finally, the simultaneous increase of CPT1A, CACT and CrAT is fundamental for PCCs to sustain FA oxidation in the presence of heavy lipid load on prostate cancer mitochondria. Indeed, the downregulation of only one of these proteins reduces PCCs metabolic flexibility with the accumulation of FA-intermediate metabolites in the mitochondria. Together, our data implicate carnitine cycle as a primary regulator of adaptive metabolic reprogramming in PCCs and suggest new potential druggable pathways for prevention and treatment of prostate cancer.
Asunto(s)
Carnitina O-Palmitoiltransferasa/genética , Proteínas de Transporte de Membrana/genética , MicroARNs/genética , Neoplasias de la Próstata/genética , Carcinogénesis/genética , Carnitina/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Oxidación-Reducción , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
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
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
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.
Asunto(s)
Senescencia Celular/genética , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Regulación de la Expresión Génica/genética , Células Madre Mesenquimatosas/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Apoptosis/genética , Western Blotting , ADN Helicasas/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Proteínas Nucleares/genética , Receptor Cross-Talk/fisiología , Proteína p130 Similar a la del Retinoblastoma/genética , Proteína p130 Similar a la del Retinoblastoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transducción Genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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
Vascular injury aimed at stenosis removal induces local reactions often leading to restenosis. The aim of this study was a concerted transcriptomic-proteomics analysis of molecular variations in a model of rat carotid arteriotomy, to dissect the molecular pathways triggered by vascular surgical injury and to identify new potential anti-restenosis targets. RNA and proteins extracted from inbred Wistar Kyoro (WKY) rat carotids harvested 4 hrs, 48 hrs and 7 days after arteriotomy were analysed by Affymetrix rat microarrays and by bidimensional electrophoresis followed by liquid chromatography and tandem mass spectrometry, using as reference the RNA and the proteins extracted from uninjured rat carotids. Results were classified according to their biological function, and the most significant Kyoro Encyclopedia of Genes and Genomes (KEGG) pathways were identified. A total of 1163 mRNAs were differentially regulated in arteriotomy-injured carotids 4 hrs, 48 hrs and 7 days after injury (P < 0.0001, fold-change > or =2), while 48 spots exhibited significant changes after carotid arteriotomy (P < 0.05, fold-change > or =2). Among them, 16 spots were successfully identified and resulted to correspond to a set of 19 proteins. mRNAs were mainly involved in signal transduction, oxidative stress/inflammation and remodelling, including many new potential targets for limitation of surgically induced (re)stenosis (e.g. Arginase I, Kruppel like factors). Proteome analysis confirmed and extended the microrarray data, revealing time-dependent post-translational modifications of Hsp27, haptoglobin and contrapsin-like protease inhibitor 6, and the differential expression of proteins mainly involved in contractility. Transcriptomic and proteomic methods revealed functional categories with different preferences, related to the experimental sensitivity and to mechanisms of regulation. The comparative analysis revealed correlation between transcriptional and translational expression for 47% of identified proteins. Exceptions from this correlation confirm the complementarities of these approaches.
Asunto(s)
Arterias Carótidas/cirugía , Estenosis Carotídea/cirugía , Perfilación de la Expresión Génica , Proteómica/métodos , Transcripción Genética , Animales , Arterias Carótidas/metabolismo , Estenosis Carotídea/metabolismo , Masculino , Ratas , Ratas Endogámicas WKYRESUMEN
Recent evidence has shown that vascular function depends not only on cells within the vessels, but is also significantly modulated by circulating cells derived from the bone marrow. A number of studies indicate that an early reendothelialization by circulating endothelial precursors after vascular injury prevents excessive cell proliferation and restenosis. Conversely, other studies concluded that the homing of other cell fractions, consisting mainly of smooth muscle precursors, cause pathological remodelling. Different cell types have been identified and characterized so far as circulating precursors able to participate in vascular repair by homing and differentiating towards endothelial cells or smooth muscle cells. Among these, endothelial precursor cells, smooth muscle progenitor cells, mesenchymal stem cells and others have been described. The origins, the hierarchy, the role and the markers of these different cell populations are still controversial. Nevertheless, different strategies have been developed so far in animal models to induce the mobilization and the recruitment of stem cells to the injury site, based on physical training, hormone injection and application of stem cell-capturing coated stents. It should also be mentioned that the limited data currently available derived from clinical trials provide contrasting results about the effective role of vascular cell precursors in restenosis prevention, thus indicating that conclusions derived from studies in animal models cannot always be directly applied to humans and that caution should be used in the manipulation of circulating progenitor cells for therapeutic strategies.
Asunto(s)
Arteriopatías Oclusivas/fisiopatología , Endotelio Vascular/fisiopatología , Músculo Liso Vascular/fisiopatología , Células Madre/patología , Animales , Arteriopatías Oclusivas/etiología , Arteriopatías Oclusivas/patología , Arteriopatías Oclusivas/cirugía , Células de la Médula Ósea/patología , Diferenciación Celular , Fusión Celular , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Constricción Patológica/patología , Constricción Patológica/fisiopatología , Células Endoteliales/patología , Endotelio Vascular/patología , Epigénesis Genética , Humanos , Células Madre Mesenquimatosas/patología , Monocitos/patología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Prevención Secundaria , Trasplante de Células Madre/métodos , Procedimientos Quirúrgicos Vasculares/efectos adversosRESUMEN
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
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
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
BACKGROUND AND AIMS: Ulcerative colitis (UC) is characterised by refractory inflammatory ulceration and damage to the colon. The mechanisms underlying impaired healing have yet to be defined. As transglutaminase expression resulting in matrix protein cross linking is associated with increased wound healing in a rat model of colitis, we hypothesised that different types of transglutaminase might also play a role in UC. PATIENTS AND METHODS: Endoscopic and histological indices were studied in 26 patients with UC (10 active and 16 inactive) and in 20 normal controls undergoing colonoscopy. Transglutaminase activity was evaluated in plasma (factor XIIIa) by a radioenzymatic method. Factor XIIIa, tissue and keratinocyte transglutaminase protein content, and mRNA expression in the colon were evaluated by western blot analysis and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Colonic location of transglutaminases and their reaction products, the epsilon-(gamma-glutamyl)lysine bonds, was evaluated by immunohistochemistry using specific monoclonal antibodies. RESULTS: Transglutaminase activity was significantly lower in the plasma of patients with active UC (4.2 (2.4) mU/ml; p<0.05 v controls) than in those with inactive UC and controls (10.6 (2.2) and 12.1 (1.7) mU/ml). As shown by western blot, protein levels of tissue transglutaminase and factor XIIIa were unchanged in active UC compared with inactive disease and controls, while the keratinocyte form was reduced in active UC. Tissue transglutaminase and factor XIIIa immunostaining was strongly present in damaged areas colocalising with isopeptide bonds. In contrast, the keratinocyte form was almost absent in active UC and localised in the upper part of the crypts in normal subjects. RT-PCR showed upregulation of tissue transglutaminase mRNA in active UC (320% compared with controls) while keratinocyte transglutaminase gene expression was downregulated in active UC. CONCLUSIONS: The results of the present study support the concept that, in the damaged colon, transglutaminases are needed in response to chronic injury and underline the key role of these enzymes in mucosal healing.
Asunto(s)
Colitis Ulcerosa/enzimología , Transglutaminasas/metabolismo , Adulto , Anciano , Western Blotting , Colitis Ulcerosa/sangre , Factor XIIIa/metabolismo , Femenino , Humanos , Mucosa Intestinal/enzimología , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Índice de Severidad de la Enfermedad , Transglutaminasas/genética , Regulación hacia Arriba , Cicatrización de HeridasRESUMEN
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
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
Investigation into DNA from archeological remains offers an inestimable tool for unraveling the history of humankind. However, a series of basic and technical difficulties renders the analysis of ancient DNA (aDNA) molecules troublesome, depending either on their own peculiar characteristics or on the complexity of processes affecting the bone matrix over time, all compromising the preservation of ancient DNA. This review underlines the contribution of many different disciplines, in particular molecular biology and genetics, to overcome these obstacles. The role of each expertise is illustrated to appropriately address the questions arising in aDNA investigations.
Asunto(s)
ADN , Fósiles , Genética , Comunicación Interdisciplinaria , Biología Molecular , Animales , Arqueología , ADN/aislamiento & purificación , Daño del ADN , Reparación del ADN , ADN Mitocondrial/genética , Amplificación de Genes , Humanos , Filogenia , CienciaRESUMEN
DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals-donkeys, horses, or crossbreeds-were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth.
Asunto(s)
ADN Mitocondrial/genética , ADN/análisis , Equidae/genética , Filogenia , Análisis de Secuencia de ADN , Animales , Secuencia de Bases , Evolución Molecular , Historia Antigua , Italia , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Neural stem cells (NSCs) could be very useful for the "cell therapy" treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF-responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF-containing medium, cells were plated on poly-L-ornithine-coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the "p53 network," was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Separación Celular/métodos , Factor de Crecimiento Epidérmico/farmacología , Proteínas del Tejido Nervioso , Proteínas , Trasplante de Células Madre/métodos , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Proteínas Sanguíneas/efectos de los fármacos , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteínas de Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/efectos de los fármacos , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Nestina , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Proteína de Retinoblastoma/efectos de los fármacos , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma , Células Madre/citología , Células Madre/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/efectos de los fármacos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The expression profiles of genes involved in cell proliferation, differentiation and programmed death were investigated in carotids of spontaneously hypertensive rats (SHR) treated with a model of surgical injury that mimics events occurring during arterial grafts, endarterectomy and organ transplantation. The mRNA level of the c-myc, angiotensin II receptor 1 (AT1), Rb/p105, Rb2/p130, Bcl-2 and Bax-alpha genes was assessed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique at different times up to 48 h after injury, while the morphological changes were evaluated 30 days after injury. The proliferation marker c-myc increases almost immediately, peaks after 4 h and returns to basal levels after 24 h; the AT1 receptor mRNA reaches its maximal level 48 h after injury. The level of cell cycle exit markers Rb/p105 and Rb2/p130 gradually decreases after injury. The apoptosis marker Bcl-2/Bax-alpha ratio shows a significant reduction only 4 h after injury, resuming the initial value after 24 and 48 h. Morphological analysis reveals that surgical injury in SHR induces adventitial and medial constrictive remodeling changes rather than intima proliferation as in balloon angioplasty. Both molecular and histological data show substantial differences with respect to normotensive rats.