RESUMEN
In this study, we evaluated the growth performance and antioxidant status of broiler chicken supplemented with the edible mushroom Agaricus bisporus. Ninety 1-d-old female broiler chickens randomly allotted to 3 dietary treatments were given either a nutritionally balanced basal diet or the basal diet supplemented with 10 or 20 g of dried mushroom/kg of feed for 6 wk on an ad libitum basis. Body weight, feed intake, and feed conversion ratio values were monitored weekly. To evaluate the antioxidant status of broiler chicken, refrigerated liver, breast, and thigh tissues were assayed for levels of glutathione, reduced glutathione, glutathione reductase, glutathione peroxidase, and glutathione S-transferase, as well as malondialdehyde at 6 wk of age. Results showed that dietary mushroom supplementation at both inclusion levels was accepted well by the broiler chicken and improved feed efficiency compared with the control diet. Dietary mushroom inclusion at 20 g/kg improved both growth performance and feed efficiency compared with control diet at 42 d of age. Dietary mushroom at both inclusion levels reduced malondialdehyde production in liver, breast, and thigh tissues and elevated glutathione peroxidase, reduced glutathione, glutathione reductase, and glutathione S-transferase compared with the control treatment, the effects being dose-dependent. These results suggest that A. bisporus mushroom exerts both a growth-promoting and tissue antioxidant-protective activity when supplemented in broiler chicken diets.
Asunto(s)
Agaricus , Alimentación Animal/análisis , Antioxidantes/metabolismo , Pollos/fisiología , Dieta/veterinaria , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Antioxidantes/química , Suplementos Dietéticos , Ingestión de Alimentos , Femenino , Hígado/química , Hígado/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Aumento de PesoRESUMEN
In an effort to elucidate the interactions between cells in the developing cortex and their microenvironment, we have employed dissociated cell cultures and immunocytochemistry to analyze the effect of collagen type IV (COL) on the proliferation and differentiation of rat cortical progenitor cells during the period of corticogenesis. COL, present in the proliferative zones throughout the period of neurogenesis, belongs to a group of macromolecular proteins that make up a considerable portion of the extracellular matrix (ECM). We have shown that this ECM molecule inhibits cell proliferation and glial cell differentiation while promoting neuronal differentiation. We have also demonstrated that COL, when applied to the cultures with basic fibroblast growth factor (bFGF), induces glial cell differentiation while continuing to promote neuronal differentiation. These results indicate that cortical progenitor cells respond differentially to local environmental signals, and that components of the ECM are involved in the regulation of corticogenesis.
Asunto(s)
Astrocitos/citología , Diferenciación Celular/efectos de los fármacos , Corteza Cerebral/citología , Colágeno/farmacología , Proteínas del Tejido Nervioso , Neuronas/citología , Células Madre/citología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/embriología , Colágeno/fisiología , Matriz Extracelular/fisiología , Feto , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/análisis , Proteínas Asociadas a Microtúbulos/análisis , Nestina , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Células Madre/efectos de los fármacosRESUMEN
Murine erythroleukemia (MEL) cells have been used as a suitable model system for studying cellular and molecular mechanisms of erythroid differentiation. In an effort to isolate and characterize genes whose expression change during differentiation, we cloned and sequenced a cDNA of 715 bp (rpS5) from a MEL cDNA library. The cloned mouse cDNA showed significant degree of structural homology in both DNA and protein level to known human and rat genes that encode the S5 proteins of 40S ribosomal subunit. The use of 715-bp cDNA as probe revealed the presence of an RNA transcript in the cytoplasm of MEL and human neuroectodermal RD/TE-671 cells. The steady-state accumulation level of this RNA transcript decreased upon induction of differentiation of both cell lines by treatment with DMSO and UDP-4, two structurally different inducers. Blockade of MEL cell differentiation by the inhibitor N6-methyladenosine preserved the constitutive expression of the rpS5 gene. DNA methylation analysis at CCGG sites located at the rpS5 gene locus in undifferentiated and differentiated MEL cells revealed that the suppression of the rpS5 gene during MEL cell differentiation is not related to any change in methylation at these sites. Moreover, the rpS5 gene continued to be expressed in cells undergoing serum-deprived apoptosis, like in control untreated cells. Therefore, we conclude that there may be a disparate pattern of expression of the rpS5 gene in differentiating and apoptotic cells. These data can be valuable in understanding the role of ribosomal proteins during differentiation and cell death (apoptosis) of neoplastic cells, although there is no experimental evidence that the suppression of the rpS5 gene is related mechanistically to the induction of differentiation. It may well be considered as part of the differentiation process.
Asunto(s)
Apoptosis/genética , Eritropoyesis/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular/genética , Clonación Molecular , Metilación de ADN , ADN Complementario/análisis , Regulación hacia Abajo , Humanos , Leucemia Eritroblástica Aguda , Ratones , Datos de Secuencia Molecular , Ratas , Células Tumorales CultivadasRESUMEN
Calretinin-expressing neurons are some of the earliest postmitotic cells to appear in the developing cerebral cortex. Lineage studies have shown that the expression of this calcium-binding protein in cortical neurons is not genetically programmed and is likely to be induced by external factors. A number of studies have clearly shown that basic fibroblast growth factor (bFGF) and a number of neurotrophins promote the proliferation and differentiation of cortical progenitor cells to a particular lineage. Here, using a culture system of dissociated rat cortical cells, we found that brain-derived neurotrophic factor and neurotrophin-3 promoted the morphological differentiation of one of the calretinin-containing neuronal subpopulations, the Cajal-Retzius cells. Another subpopulation of calretinin-expressing cells of smaller size and bipolar form was generated when cultures were treated with bFGF. The progenitors of these neurons were stimulated by bFGF to divide a number of times before initiating their differentiation programme. The number of calretinin-expressing neurons increased further when cultures were treated with a combination of bFGF and retinoic acid.
Asunto(s)
Corteza Cerebelosa/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas del Tejido Nervioso/análisis , Neuronas/efectos de los fármacos , Proteína G de Unión al Calcio S100/análisis , Animales , Calbindina 2 , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebelosa/química , Corteza Cerebelosa/citología , Neuronas/química , Neuronas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Lineage studies have recently shown that the expression of calcium-binding proteins in neurons of the cerebral cortex is not genetically programmed and is likely to be induced by external factors. Current hypotheses suggest that basic fibroblast growth factor (bFGF) and a number of neurotrophins play important roles in the proliferation and differentiation of cortical progenitor cells to a particular lineage. Using a dissociated cell culture system, we found that bFGF and the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor differentially affect the expression of the calcium-binding protein calbindin in selective neuronal subpopulations in the developing cerebral cortex. Specifically, BDNF and NT-3 greatly promoted the morphological differentiation of a relatively small, early-generated population of GABAergic neurons and induced the expression of calbindin in these cells. Furthermore, treatment with BDNF, NT-3, and bFGF produced an two- to threefold increase in the number of newly generated calbindin-positive neurons. The effect of bFGF was more striking in earlier (E14) than later (E16) ages, whereas the action of neurotrophins was independent of the age from which the cultures were prepared. Switching experiments combined with BrdU incorporation have suggested that NT-3 acts on postmitotic neurons rather than on proliferating progenitors to induce calbindin expression and that its action is mediated via trk receptors. Application of retroviral vectors in culture resulted in the presence of neuronal clones that were predominantly heterogeneous with regard to calbindin expression, suggesting, in agreement with our earlier in vivo studies, that the expression of this calcium-binding protein is not lineage dependent. Our results characterize the roles of BDNF, NT-3, and bFGF in the expression of calbindin in developing neocortical neurons.
Asunto(s)
Corteza Cerebral/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/análisis , Neuronas/efectos de los fármacos , Proteína G de Unión al Calcio S100/análisis , Animales , Astrocitos/fisiología , Calbindinas , Diferenciación Celular/efectos de los fármacos , División Celular , Linaje de la Célula , Células Cultivadas , Senescencia Celular , Corteza Cerebral/embriología , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Neuronas/clasificación , Neuronas/citología , Neurotrofina 3 , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Proteína G de Unión al Calcio S100/biosíntesis , Proteína G de Unión al Calcio S100/genética , Transfección , Ácido gamma-Aminobutírico/análisisRESUMEN
A series of ureido derivatives of pyridine (UDPs) were developed as inducers of leukemic cell differentiation. Fifteen agents prepared by coupling aminopyridines with appropriate isocyanates were structurally identified and tested for both antiproliferative and differentiation inducing activity in cultures of murine and human leukemic cells. Five of these lipophilic compounds [2-(3-ethylureido)-pyridine (1), 2-(3-ethylureido)-6-methylpyridine (4), 2,6-bis-(3-ethylureido)-pyridine (7), 2-(3-ethylureido)-5-methylpyridine (14) and 2-(3-ethylureido)-4,5-dimethylpyridine (15)], promoted terminal erythroid maturation of murine erythroleukemia cells (MEL) (95% hemoglobin producing cells) and stimulated hemoglobin synthesis at concentrations as low as 0.075-0.5 mM. These concentrations are 50-70 fold lower than the optimum inducing concentration of hexamethylene bisacetamide (HMBA), a potent known inducer of differentiation. The proportion of cells induced by each ureido derivative of pyridine was concentration-dependent. Moderate inhibition of cell growth was obtained by these agents at optimum inducing concentrations. Agent 1 also stimulated hemoglobin synthesis in 18% of human erythroleukemia K-562 cells and promoted granulocytic differentiation in 24% of human promyelocytic leukemia HL-60 cells. Structure-activity relationships indicate that 7 was the most potent inducer of all UDPs which contain an ethylureido group attached at 2 position of the pyridine ring as a major structural feature needed for inducing activity. These findings indicate that the ureido derivatives of pyridine are a new class of inducers of murine erythroleukemia cells and, to a lesser extent of granulocytic differentiation, of leukemic HL-60 cells in vitro.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Leucemia Eritroblástica Aguda/patología , Piridinas/farmacología , Urea/análogos & derivados , Animales , Ciclo Celular , Leucemia Promielocítica Aguda/patología , Ratones , Relación Estructura-Actividad , Células Tumorales Cultivadas/efectos de los fármacos , Urea/farmacologíaRESUMEN
In a previous study we reported that ribosomal protein S5 gene is suppressed in differentiating and not in proliferating or apoptotic murine erythroleukaemia (MEL) cells (Vizirianakis et al., 1999). We wish to report here the isolation, characterisation and expression of the full length cDNA for another ribosomal protein, the L35a (rpL35a), in MEL cells. This cDNA shares significant structural homology in both DNA and protein levels to genes encoding the rat and human L35a ribosomal proteins. Northern blot hybridisation analysis has shown that the steady-state level of rpL35a mRNA is progressively reduced during differentiation of MEL cells along the erythrocytic maturation pathway induced by DMSO or UDP-4, two structurally unrelated inducers of differentiation. However, in cells where differentiation was inhibited by N(6)-methyladenosine, the level of rpL35a RNA transcripts was not affected. In addition, rpL35a gene expression was not altered in apoptotic MEL cells. Furthermore, the suppression of L35a gene was not correlated to any change in DNA methylation at CCGG sites located at the rpL35a gene locus in undifferentiated and differentiated MEL cells, as we observed for the rpS5 gene. Overall, these data suggest that the expression of ribosomal genes, the L35a of 60S ribosomal subunit and the S5 of 40S ribosomal subunit, are regulated by a common mechanism in differentiating MEL cells, leading to the observed decrease in ribosomal function.
Asunto(s)
Leucemia Eritroblástica Aguda/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Clonación Molecular , ADN Complementario , Dimetilsulfóxido/farmacología , Leucemia Eritroblástica Aguda/genética , Ratones , Datos de Secuencia Molecular , Piridinas/farmacología , ARN Neoplásico/biosíntesis , Transcripción Genética , Células Tumorales Cultivadas , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Recent studies have localized gamma-aminobutyric acid (GABA)-containing neurons and identified cells that express subunits of the GABAA receptor in the proliferative zone of the developing cerebral cortex and have demonstrated a role for GABA in cortical neurogenesis. We examined here the interactions between a number of neurotrophic factors, known to be involved in cortical cell proliferation and differentiation, and the GABAergic system (GABA and GABAA receptors) in the regulation of cell production in dissociated cortical cell cultures. We found that basic fibroblast growth factor (bFGF) increased the number of cells labelled for the alpha 1 subunit of the GABAA receptor but not for the alpha 2, alpha 3 or alpha 5 subunits. The alpha 1 subunit was expressed by the majority of proliferating neuroepithelial cells as well as by differentiated neurons. We also found that activation of the GABAA receptor by GABA or muscimol inhibited the proliferative effects of bFGF on cortical progenitors, leading to an increased number of differentiated neurons. These results suggest that bFGF stimulates cell proliferation and GABAA receptor expression in cultured progenitor cells of the developing neocortex, and that GABA regulates cell production by providing a feedback signal that terminates cell division.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Agonistas de Receptores de GABA-A , Factores de Crecimiento Nervioso/farmacología , Células Madre/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología , Animales , Recuento de Células/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Neuronas/citología , Neuronas/efectos de los fármacos , RatasRESUMEN
The appearance of 5-hydroxytryptamine (serotonin; 5-HT) in the cerebral cortex coincides with developmental events such as cell proliferation, survival, and differentiation. We tested the hypothesis that 5-HT plays a role in these events by examining rat cortical progenitor cells in vitro. Using bromodeoxyuridine incorporation we found that 5-HT did not affect the proliferation of these cells, but a cell survival assay indicated that it promoted their survival. The observed survival effect was mimicked by the 5-HT2a/2c receptor agonist alpha-methyl-5-HT and blocked by the 5-HT2a receptor antagonist cinanserin. Consistent with increased survival was the finding, using the terminal transferase nick end labeling method, of reduced cell death in cultures exposed to 5-HT. Immunohistochemical analysis with cell-specific markers revealed that the effect of 5-HT was directed specifically to the glutamate-containing neuronal population and not to any other cortical cell types. These results indicate that 5-HT does not exert its effects on dividing neuroepithelial cells in the developing cortex, but rather on postmitotic neurons.
Asunto(s)
Corteza Cerebral/citología , Ácido Glutámico/análisis , Neuronas/efectos de los fármacos , Receptores de Serotonina/efectos de los fármacos , Serotonina/farmacología , Células Madre/citología , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/embriología , Cinanserina/farmacología , Neuronas/química , Neuronas/clasificación , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Receptores de Serotonina/fisiología , Serotonina/análogos & derivadosRESUMEN
Basic fibroblast growth factor (bFGF) has been shown to influence the survival, proliferation and differentiation of a variety of cell types in the nervous system. In this investigation we have examined the action of bFGF on: (i) the rate of proliferation; (ii) cell cycle parameters; (iii) the maintenance of cell division; (iv) the recruitment of quiescent cells; and (v) the degree of differentiation of cortical progenitor cells in cultures prepared from E16 rat embryos. The proliferation rate (labelling index) of cortical progenitor cells doubled in the presence of bFGF over 48 h. However, the lengths of the cell cycle phases were unchanged. Clones marked with a recombinant retrovirus on the first day in vitro (DIV) grew significantly larger in the presence of bFGF. Furthermore, many of the clones examined in control cultures had ceased to divide after a maximum of four cell cycles, whereas almost all clonally related cells were still dividing in the presence of bFGF 4 days later, i.e. for at least six cell cycles. Basic FGF also stimulated the division of quiescent progenitor cells, which otherwise would have differentiated or undergone cell death. The degree of neuronal and glial differentiation was studied after 5 DIV using MAP-2 and GFAP immunocytochemistry. In the presence of bFGF, the percentage of MAP-2-labelled cells was less than half that of control cultures, whereas the number of cells immunoreactive for nestin (a marker of progenitor cells) remained very high. Cells immunoreactive for GFAP were present in bFGF-treated cultures, yet were extremely rare in control conditions. These experiments show that bFGF, a potent mitogen for cortical progenitor cells, has no effects on the parameters of their cell cycle but extends their proliferative capability, promotes their survival and delays their differentiation into neurons.
Asunto(s)
Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre/efectos de los fármacos , Animales , Células Cultivadas/efectos de los fármacos , Técnicas In Vitro , Ratas , Ratas Sprague-DawleyRESUMEN
Human TE-671 cells have been used to study several aspects of neuroectodermal tumors in culture. Since the human TE-671 cell lines has been re-identified as a rhabdomyosarcoma (RD) rather than a medulloblastoma due to the presence of muscle-type nicotinic acetylcholine receptors, we re-investigated the nature of RD/TE-671 cells and characterized their differentiation induced by 2-(3-ethylureido)-6-methylpyridine (UDP-4), a potent inducer of differentiation of neoplastic cells. RD cells were also used for comparative studies. RD/TE-671 cells exposed to UDP-4 were differentiated irreversibly into postmitotic cells expressing mainly neurofilaments and, to a lesser extent, myoid proteins. In contrast to RD cells that expressed preferentially myoid and not neurofilament proteins (NFPs) upon treatment with UDP-4, differentiated RD/TE-671 cells exhibited characteristic dendritic processes and expressed NFPs (NFP68, NFP160, and NFP200), parvalbumin (calcium-binding protein), and neuron-specific enolase, as well as a small amount of vimentin and desmin. In addition, differentiated RD/TE-671 cells expressed memory for differentiation and underwent an irreversible limitation of proliferation, loss of clonogenic potential, selective repression of c-myc and p53 proto-oncogenes, and changes in cell surface architecture. Treatment of RD/ TE-671 cells with nerve growth factor or epidermal growth factor in the presence of UDP-4 did not alter the phenotype of differentiated cells, whereas co-treatment with 12-O-tetradecanoylphorbol-13-acetate and UDP-4 enhanced morphological differentiation. Therefore, we conclude that: (a) RD/TE-671 cells challenged with UDP-4 express memory to differentiate in the absence of inducer; (b) in contrast to RD cells, RD/TE-671 cells appear to be multipotent cells of neuroectodermal origin capable of differentiation into cells expressing neuronal rather than myoid proteins upon treatment with UDP-4; and (c) differentiation of RD/TE-671 cells leads to selective cessation of cell proliferation and repression of c-myc and p53 proto-oncogenes.