RESUMEN
AIM: The aim of the present study was to investigate heart rate (HR), salivary cortisol (sC) alpha-amylase (sAA) and rating of perceived exertion (RPE) in relation to competition outcome during a half marathon. METHODS: HR was monitored and salivary samples were collected during an official half marathon in five Master endurance runners (age 47 ± 7 years). RPE was collected using a 100-mm Visual Analogue Scale (VAS) 30 minutes after the end of competition. RESULTS: Performance corresponded to 94% of their personal best (PB). Athletes spent 53.7% of total race time at intensities above 95% HRmax. RPE showed values of 68 ± 8 mm. With respect to pre-competition values (25.54 ± 6.39 nmol/L), sC concentrations significantly increased (P=0.043) by 59% immediately after the race (40.54 ± 3.95 nmol/L) and remained elevated until 1 h post exercise. Pre-competition sAA concentrations (90.59 ± 42.86 U/mL) were 118% higher (P=0.043) with respect to time-matched baseline values (197.92 ± 132 U/mL). sAA increased (192%; P=0.043) immediately after the race and was higher than time-matched resting samples. The better each athlete performed the greater cortisol increase during exercise (P<0.001). Performance was not correlated to the anticipatory sAA (the percent difference between pre-competition values and time-matched baseline ones) or to the sAA increase during exercise. CONCLUSION: This is the first attempt to study the stress-related responses during official endurance competitions in master runners. Although the strict criteria of inclusion might have limited the statistical significance, the present findings indicate that endurance competition is a remarkable stressor for psycho-physiological aspects of master athletes.
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Frecuencia Cardíaca , Hidrocortisona/análisis , Esfuerzo Físico , Carrera , Saliva/química , alfa-Amilasas/análisis , Atletas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resistencia FísicaRESUMEN
The aim of this study was to evaluate the effects of an official Taekwondo competition on the heart rate (HR), salivary α-amylase (sA-A), salivary free cortisol (sC), and Profile of Mood States (POMS) in 10 young male (14±0 years) and six female (13±1 years) athletes. POMS and hormones were measured 15 min before and directly after the competition. During the recovery phase (30 and 90 min), sA-A and sC were also measured. HR measured during the competition was expressed as a percentage of individual's maximal heart rate (%HR(max) ) to evaluate the intensity of exercise. During the competition, athletes spent 65% of the time working at HR>90% of individuals HR(max). A significant increase (P<0.0001) in sA-A (115%) was observed at the end of the match. At 30 min of recovery, sA-A returned to the pre-competition level. The peak sC values were observed at 30 min of recovery (P<0.001), returning to the pre-competition level at 90 min of recovery. A gender difference (P=0.01) emerged only for sC, although a similar trend was observed for female and male athletes. Significantly higher post-match scores emerged for Anger-hostility (pre: 6.1±1.1, post: 11.2±1.9; P=0.03) and Depression-dejection (pre: 4.5±0.5, post: 10.2±1.9; P=0.006), whereas the reverse picture was observed for Vigour-activity (pre: 23.2±1.2, post: 16.3±1.7; P=0.0006). Taekwondo competition results in temporary changes in the stress-related parameters measured in this study. The present findings suggest that this experimental paradigm can represent a useful model for further research on the effects of various stressors (i.e., training and competition) in Taekwondo athletes of different levels (i.e., novice, international).
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Conducta Competitiva/fisiología , Artes Marciales/fisiología , Artes Marciales/psicología , Adolescente , Amilasas/metabolismo , Análisis de Varianza , Biomarcadores/metabolismo , Femenino , Humanos , Hidrocortisona/metabolismo , MasculinoRESUMEN
Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development.
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Moléculas de Adhesión Celular Neuronal , Proteínas de la Membrana/análisis , Proteínas del Tejido Nervioso/análisis , Neuronas/citología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Adhesión Celular , Membrana Celular/análisis , Membrana Celular/metabolismo , Células Cultivadas , Mapeo Cromosómico , Contactina 1 , Contactinas , ADN/análisis , ADN/genética , Fibronectinas/análisis , Fibronectinas/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Ligamiento Genético , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Neuronas/análisis , Neuronas/metabolismo , Hibridación de Ácido Nucleico , Fosfatidilinositoles/metabolismo , Biosíntesis de Proteínas , ARN/genética , ARN/metabolismo , Receptores de Antígenos de Linfocitos B/análisis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Extractos de Tejidos/análisis , Extractos de Tejidos/genéticaRESUMEN
Many environmental signals, including ionizing radiation and UV rays, induce activation of Egr-1 gene, thus affecting cell growth and apoptosis. The paucity and the controversial knowledge about the effect of electromagnetic fields (EMF) exposure of nerve cells prompted us to investigate the bioeffects of radiofrequency (RF) radiation on SH-SY5Y neuroblastoma cells. The effect of a modulated RF field of 900 MHz, generated by a wire patch cell (WPC) antenna exposure system on Egr-1 gene expression, was studied as a function of time. Short-term exposures induced a transient increase in Egr-1 mRNA level paralleled with activation of the MAPK subtypes ERK1/2 and SAPK/JNK. The effects of RF radiations on cell growth rate and apoptosis were also studied. Exposure to RF radiation had an anti-proliferative activity in SH-SY5Y cells with a significant effect observed at 24 h. RF radiation impaired cell cycle progression, reaching a significant G2-M arrest. In addition, the appearance of the sub-G1 peak, a hallmark of apoptosis, was highlighted after a 24-h exposure, together with a significant decrease in mRNA levels of Bcl-2 and survivin genes, both interfering with signaling between G2-M arrest and apoptosis. Our results provide evidence that exposure to a 900 MHz-modulated RF radiation affect both Egr-1 gene expression and cell regulatory functions, involving apoptosis inhibitors like Bcl-2 and survivin, thus providing important insights into a potentially broad mechanism for controlling in vitro cell viability.
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Ciclo Celular/efectos de la radiación , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Expresión Génica/efectos de la radiación , Microondas/efectos adversos , Neuroblastoma/patología , Apoptosis/efectos de la radiación , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Citometría de Flujo , Humanos , Neuroblastoma/metabolismo , Propidio/metabolismo , Dosis de Radiación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIM: The aim of the present study was to investigate the stress--related changes of a TeamGym competition considering both physiological [i.e. salivary cortisol (sC) and alpha--amylase (sAA)] and psychological (i.e. state anxiety) responses in relation to exercise intensity and competition outcomes. METHODS: Eleven (5 males and 6 females) elite TeamGym athletes (age: 21--28 yrs) were administered the State--Trait Anxiety Inventory before an official international TeamGym competition. sAA and sC samples were collected 15 minutes prior to competition, after each apparatus, 10--min and 30--min after competition. Exercise intensity was estimated by heart rate (HR) recording and performance was evaluated by three international judges. All these parameters were correlated with competition outcomes. RESULTS: TeamGym competition posed a low exercise load (most of exercise was performed below 85% of the individual HR max ). Significant increases (P<0.004) in sAA (3.53 fold induction) and state anxiety (P=0.045) were observed, with respect to baseline values. Conversely, sC remained stable throughout the competition. Significant (P=0.029) correlation between sAA, state anxiety and competition outcomes emerged. CONCLUSIONS: Present findings provide the first evidence that the psycho--physiological stress response prior to and during competition can affect performance outcome, especially in a technical sport such as TeamGym.
RESUMEN
The transcription factor cAMP response element binding protein 2 (CREB2) belongs to a family of proteins containing a basic region as DNA-binding domain and a leucine zipper as a dimerization domain in its C-terminus. Using indirect immunofluorescence labeling of cells we show that CREB2 is a nuclear protein. To identify the signal(s) required for nuclear targeting of CREB2, various domains of the protein were expressed in COS cells as fusion proteins with glutathione S-transferase and their cellular location assayed by indirect immunofluorescence using antibodies directed against the glutathione S-transferase moiety of the fusion proteins. The results show that the nuclear targeting signal is located in the C-terminal part of the molecule. Deletion mutagenesis revealed that the basic region of CREB2, encompassing amino acids 280 to 300, is sufficient for sorting CREB2 to the nucleus. Single point mutations of basic amino acids within the basic region of CREB2 identified the sequence KKLKK (amino acids 280 to 284) as important for nuclear targeting. Thus, the basic region of CREB2 is necessary not only for tethering CREB2 to DNA but also for sorting CREB2 to the nucleus. However, sequences outside the basic region are additionally required for efficient nuclear sorting of CREB2.
Asunto(s)
Núcleo Celular/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Membrana Nuclear/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 4 , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Western Blotting , Células COS , Glutatión Transferasa/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Corticotropin-releasing factor (CRF) and vasoactive intestinal polypeptide (VIP) are neuropeptides displaying a variety of short-term effects in the nervous system. It is shown here in transfection experiments of an immortalized noradrenergic locus coeruleus-like cell line that both CRF and VIP also trigger a signaling cascade capable of activating gene transcription. To elucidate the signaling pathway leading to transcriptional induction, cells were transfected with an inhibitor for cAMP-dependent protein kinase, targeted to the nucleus via a nuclear-localization signal. Transcriptional induction of a reporter gene by CRF and VIP was blocked in these cells, indicating that the cAMP-dependent protein kinase is required for transducing CRF and VIP generated signals into the nucleus. Additionally, transfection experiments with a reporter gene containing cAMP response elements in its regulatory region demonstrate that CRF and VIP receptor activation induce transcription through this genetic regulatory element. We conclude that long-term effects of CRF and VIP in neurons are likely to be mediated by the transcriptional regulation of CRF and VIP-responsive genes via the cAMP signaling pathway.
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Hormona Liberadora de Corticotropina/fisiología , AMP Cíclico/metabolismo , Neuronas/metabolismo , Transducción de Señal , Transcripción Genética/fisiología , Péptido Intestinal Vasoactivo/fisiología , Animales , Secuencia de Bases , Línea Celular Transformada , Núcleo Celular/enzimología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Cartilla de ADN , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Humanos , Norepinefrina/fisiología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The transcription factor cAMP-response element binding protein 2 (CREB2), a member of the family of basic region leucine zipper proteins, has been suggested to function in the brain as a repressor of long-term memory. Using recombinant proteins we show that CREB2 binds in vitro to the palindromic cAMP response element derived from the secretogranin II gene. Recent studies of the chromogranin B, secretogranin II and enkephalin genes showed that CREB2 functioned as a repressor of cAMP-induced transcription. We analyzed the ability of CREB2 to repress transcription using model promoters. A molecular dissection of the CREB2 molecule revealed that CREB2 lacks a transferable repressor domain suggesting that CREB2 may function solely as a "passive" transcriptional repressor. In contrast, "active" repressor domains derived from the thyroid hormone receptor alpha or the NK10 zinc finger protein containing a "Krüppel associated box" could be transfered to a heterologous DNA-binding domain and functioned as fusion proteins in repressing transcription of a reporter gene. In addition, a strong activation domain located at the N-terminus was identified in the CREB2 protein suggesting that CREB2 may act as an activator of transcription by binding to different genetic regulatory elements.
Asunto(s)
Factores de Transcripción/química , Factor de Transcripción Activador 4 , Glutatión Transferasa/química , Humanos , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Células Tumorales CultivadasRESUMEN
Cerebrospinal fluid (CSF)/serum concentration gradients (Q) of individual proteins (albumin, IgG, alpha 2-macroglobulin) have been studied in controls and in patients in whom the lumbar CSF flow is altered (medullary compression) or the blood-CSF barrier (BCB) function impaired (acute idiopathic polyneuropathy and acute meningoencephalitis). The analysis of relationships among protein Q has been performed by total and multiple regressions and the actual BCB permeability to individual proteins has been interpreted according to the accepted theoretical porous or vesicular BCB models. The exponential Q-IgG vs. Q-albumin total regression, and the poor Q-alpha 2-macroglobulin vs. Q-albumin regression found in controls, together with the different multiple regressions among proteins and the high Q-IgG vs. Q-albumin partial regression coefficients found in medullary compression, acute idiopathic polyneuropathy and acute meningoencephalitis, indicated that different permeability mechanisms can be postulated. Heterogeneous, fairly independent permeability BCB mechanisms maintain the normal CSF/serum protein concentration gradient. Pinocytotic vesicles or pores of radius exceeding 1000-1500 A, probably located at the capillary endothelium, account for the main serum-derived CSF protein fraction(s) with large hydrodynamic radius (R). A more selective endothelial vesicular transport with a radius of 250 A transfers a negligible amount of protein from serum into CSF. Proteins with small R also enter the CSF through a set of selective pores of radius 120 A, probably at the level of the choroidal epithelium. Pinocytotic vesicles with a radius of 250 A and increased rate of formation induce the accumulation of proteins below an obstruction of lumbar CSF flow. An increased formation rate of vesicles with a radius of 450 A can explain the increased capillary permeability in nerve roots in acute idiopathic polyneuropathy. Loss of selectivity was the main feature of BCB in acute meningoencephalitis, and it seemed to be due to pores or vesicles with a radius larger than 1000-1500 A. The heterogeneity of BCB mechanisms must be taken into account when the intrathecal synthesis of a protein, also derived from serum (for example IgG), has to be measured.
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Proteínas Sanguíneas/metabolismo , Barrera Hematoencefálica , Encefalopatías/líquido cefalorraquídeo , Permeabilidad Capilar , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Meningoencefalitis/líquido cefalorraquídeo , Pinocitosis , Polineuropatías/líquido cefalorraquídeo , Albúmina Sérica/líquido cefalorraquídeo , Compresión de la Médula Espinal/líquido cefalorraquídeo , alfa-Macroglobulinas/líquido cefalorraquídeoRESUMEN
The resting energy expenditure and the adaptation of the autonomic nervous system induced by sport activities in sedentary women and in female professional basketball players have been studied. Resting energy expenditure, body composition and the level of activity of the autonomic nervous system were measured before and after a period of six months. The physical activity induced an increase in resting energy expenditure and free fat mass without variations in body weight. Basketball players showed a significant increase in the parasympathetic activity, measured by the power spectral analysis of the heart rate variability. These findings demonstrate that resting energy expenditure is higher in the athletes than in sedentary women, despite the augmented parasympathetic activity that is usually related to lower energy expenditure.
Asunto(s)
Composición Corporal/fisiología , Metabolismo Energético/fisiología , Descanso/fisiología , Deportes/fisiología , Adulto , Sistema Nervioso Autónomo/fisiología , Peso Corporal/fisiología , Ingestión de Energía/fisiología , Femenino , Frecuencia Cardíaca/fisiología , HumanosRESUMEN
The aim of this study was to evaluate the effects of a simulated firefighting intervention on salivary alpha-amylase (sA-A), free cortisol (sC), anxiety (STAI), and profile of mood states (POMS) in 20 male firefighters (age 32 +/- 1 years, VO(2peak): 43 +/- 5 ml/kg per min). During the 12-min firefighting intervention (ambient temperature: 13 +/- 1 degrees C; relative humidity: 63 +/- 1%), individuals spent 63 +/- 28% of the time working at heart rate (HR) >85% of individual HR(max), [La] (peak) 9.2 +/- 2.9 mM and ratings of perceived exertion 16 +/- 2. At 30 min post-intervention significant (p < 0.001) increases in sA-A (174%) and sC (109%) were found with regard to values recorded before and after 90 min of the firefighting intervention. Since no differences emerged between pre-intervention and post intervention for STAI and POMS values, the hormonal changes were attributable to the intense physical stress of the simulated intervention. Further research is needed during real firefighting activities, where high emotional stress may also be present.
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Ansiedad/metabolismo , Incendios , Hidrocortisona/metabolismo , Enfermedades Profesionales/psicología , alfa-Amilasas Salivales/metabolismo , Estrés Fisiológico/fisiología , Adulto , Escalas de Valoración Psiquiátrica Breve , Estudios de Cohortes , Frecuencia Cardíaca , Humanos , Ácido Láctico/sangre , Masculino , Enfermedades Profesionales/metabolismo , Enfermedades Profesionales/fisiopatología , Resistencia FísicaRESUMEN
The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The human synapsin I gene was functionally analyzed to identify control elements directing the neuron-specific expression of synapsin I. By directly measuring the mRNA transcripts of a reporter gene, we demonstrate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between mouse and human. Deletion of a putative binding site for the zinc finger protein, neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), abolished neuron-specific expression of the reporter gene almost entirely, allowing constitutively acting elements of the promoter to direct expression in a non-tissue-specific manner. These constitutive transcriptional elements are present as a bipartite enhancer, consisting of the region upstream (nucleotides -422 to -235) and downstream (nucleotides -199 to -143) of the putative NRSF/REST-binding site. The latter contains a motif identical to the cAMP response element. Both regions are not active or are only weakly active in promoting transcription on their own and show no tissue-specific preference. From these data we conclude that neuron-specific expression of synapsin I is accomplished by a negative regulatory mechanism via the NRSF/REST binding motif.
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Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Neuronas/metabolismo , Regiones Promotoras Genéticas , Sinapsinas/biosíntesis , Sinapsinas/genética , Transcripción Genética , Animales , Secuencia de Bases , Células CHO , Secuencia Conservada , Cricetinae , Globinas/biosíntesis , Humanos , Ratones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN Mensajero/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , TATA Box , Transfección , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
A case of hypokalemic myopathy with systolic hypertension due to chronic liquorice ingestion is described. The clinical features quickly receded on potassium replacement therapy and discontinuation of liquorice ingestion. The relationship between the clinical features and the blood values and instrumental data is shown. The aldosterone-like action of the active principle of liquorice is underlined.
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Enfermedades Transmitidas por los Alimentos/complicaciones , Glycyrrhiza , Hipopotasemia/etiología , Enfermedades Musculares/etiología , Plantas Medicinales , Humanos , Hipertensión/etiología , Masculino , Persona de Mediana EdadRESUMEN
cAMP has neutrotrophic effects in the nervous system. We have investigated whether there is a correlation between cAMP-induced neurite outgrowth and induction of chromogranin B and synapsin I gene expression. These genes encode marker proteins of distinct populations of vesicles in neurons, neuroendocrine and endocrine cells, and in addition, they contain a cAMP response element (CRE) in their upstream regions, making it likely that cAMP-induced neuronal differentiation might be accompanied by increased transcription of these genes. We increased intracellular cAMP levels in neuronal and neuroendocrine cells and analyzed the levels of chromogranin B and synapsin I mRNA. Our data revealed that, while chromogranin B mRNA was in fact induced following cAMP stimulation, synapsin I mRNA was not affected. To analyze the cis-acting sequences, we constructed hybrid genes containing the upstream region of the mouse chromogranin B gene fused to a reporter gene. Similar plasmids containing the synapsin I or the glucagon promoter were constructed. Transfections of neuronal and endocrine cells, together with deletion mutagenesis, revealed that the CRE of the chromogranin B gene mediated the effect of cAMP upon transcription. This effect was mimicked by overexpression of the catalytic subunit of the cAMP-dependent protein kinase. In addition, overexpression of the negative-acting CRE-binding protein CREB-2 revealed that the chromogranin B CRE functions as a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Synapsin I gene expression, however, was not induced by either elevated intracellular cAMP concentration or by overexpression of protein kinase A, although a similar pattern of proteins, including CREB, bound to the synapsin I and chromogranin B CRE in vitro. Thus while the CRE element in the chromogranin B gene promoter is responsive to cAMP, the same element, when present in the synapsin I promoter, does not confer cAMP inducibility.
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Cromograninas/genética , AMP Cíclico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regiones Promotoras Genéticas , Proteínas Quinasas/farmacología , Sinapsinas/genética , 1-Metil-3-Isobutilxantina/farmacología , Animales , Secuencia de Bases , Bucladesina/farmacología , Línea Celular Transformada , Cromogranina B , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Elementos de Facilitación Genéticos , Expresión Génica , Ratones , Datos de Secuencia Molecular , Células PC12 , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes de Fusión , Transcripción GenéticaRESUMEN
Research on the regulation of transcription in mammals has focused in recent years mainly on the mechanism of transcriptional activation. However, transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and might play an important role in many biological processes. To understand the molecular mechanism of transcriptional repression, the activity of eight mammalian repressors or repressor domains was investigated using a set of model promoters in combination with two different transcriptional detection methods. The repressors studied were: REST, the thyroid hormone receptors alpha and beta, the zinc finger protein NK10 containing a 'krüppel-associated box' (KRAB), repressor domains derived from the proteins Egr-1, Oct2A and Dr1 and the repressor/activator protein YY1. Here we show that the repressor domains of REST, Egr-1, the thyroid hormone receptors alpha< and beta and NK10 were transferable to a heterologous DNA-binding domain and repressed transcription from proximal and distal positions. Moreover, these repressor domains also blocked the activity of a strong viral enhancer in a 'remote position'. Thus, these domains are 'general' transcriptional repressor domains. The 'krüppel-associated box' was the most powerful repressor domain tested. In contrast, the repressor domains derived from Oct2A and Dr1 were inactive when fused to a heterologous DNA-binding domain. The repressor domain of YY1 exhibited transcriptional repression activity only in one of the transcriptional assay systems. The recruitment of histone deacetylases to the proximity of the basal transcriptional apparatus was recently discussed as a mechanism for some mammalian transcriptional repressor proteins. Here we show here that histone deacetylase 2, targeted to the reporter gene via DNA-protein interaction, functions as a transcriptional repressor protein regardless of the location of its binding site within the transcription unit.
Asunto(s)
Proteínas Inmediatas-Precoces , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcripción Genética/efectos de los fármacos , Animales , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Factores de Unión al ADN Específico de las Células Eritroides , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histona Desacetilasa 2 , Histona Desacetilasas/metabolismo , Humanos , Mamíferos , Factor 2 de Transcripción de Unión a Octámeros , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Transcripción YY1RESUMEN
The activator protein 1 (AP-1) transcription factor is composed of heterodimers of the Fos/activating transcription factor (ATF) and Jun subfamilies of basic-region leucine-zipper (B-ZIP) proteins. In order to determine the identities of individual B-ZIP proteins in various AP-1 complexes we tested the effect of dominant-negative mutants to the B-ZIP proteins c-Fos, ATF2, ATF4 and CCAAT-enhancer-binding protein (C/EBP) on the activities of the collagenase and c-Jun promoters. These dominant-negative mutants inhibit DNA binding of wild-type B-ZIP proteins in a leucine-zipper-dependent fashion. Transcription of a collagenase promoter/reporter gene was induced in HepG2 hepatoma cells by expression of c-Fos and c-Jun, administration of PMA ("TPA") or by expression of a truncated form of MEK (mitogen-activated/extracellular-signal-regulated kinase kinase) kinase-1, MEKK1Delta. In all cases, the dominant-negative mutants A-Fos and A-ATF2 decreased collagenase promoter activity. However, A-ATF4 and A-C/EBP had no effect. A-Fos and A-ATF2 also reduced MEKK1Delta-induced stimulation of the c-Jun promoter. In contrast, constitutive c-Jun promoter activity was blocked solely by A-ATF2, strongly suggesting that ATF2 and/or an ATF2-dimerizing protein are of major importance for c-Jun transcription in unstimulated cells. These results demonstrate that AP-1 transcription factors of different compositions control c-jun gene transcription in resting or stimulated cells.
Asunto(s)
Colagenasas/genética , Regulación Neoplásica de la Expresión Génica , Quinasa 1 de Quinasa de Quinasa MAP , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-jun/genética , Factor de Transcripción AP-1/metabolismo , Secuencia de Aminoácidos , Carcinoma Hepatocelular , Células Cultivadas , Genes Reporteros , Genes jun , Humanos , Leucina Zippers , Neoplasias Hepáticas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factor de Transcripción AP-1/química , Factores de Transcripción/metabolismoRESUMEN
Corticotropin-releasing factor (CRF), a neuropeptide of 41 amino acids, acts as the major physiological regulator of the basal and stress-induced release of corticotropin (ACTH), beta-endorphin and other proopiomelanocortin-derived peptides from the anterior pituitary gland. In addition to its endocrine activity, CRF displays extrahypophysiotropic effects, mainly as a regulator of stress responses. We show here that CRF may additionally function as a differentiating factor in immortalized noradrenergic neuronal CATH.a cells that express CRF receptor type I and resemble locus coeruleus-derived neurons. CRF triggers morphological changes in CATH.a cells including the appearance of extended long, slender neurites with prominent growth cones. CRF-treated CATH.a cells exhibit a morphology similar to locus coeruleus neurons in primary culture. CRF-induced neurite outgrowth of CATH.a cells was blocked by addition of inhibitors for cAMP-dependent protein kinase or extracellular signal-regulated protein kinase (ERK), a subtype of the mitogen-activated protein kinases. The participation of ERK within the CRF signalling cascade was further confirmed by Western blot experiments, with antibodies directed against the phosphorylated form of ERK, and also with transcription-based assays. We conclude that CRF functions as a differentiating factor of CATH.a cells via the cAMP and the MAP kinase signalling pathways.
Asunto(s)
Hormona Liberadora de Corticotropina/farmacología , AMP Cíclico/metabolismo , Proteínas Inmediatas-Precoces , Sistema de Señalización de MAP Quinasas/fisiología , Neuritas/efectos de los fármacos , Neuronas/enzimología , Norepinefrina/genética , Proteínas Proto-Oncogénicas , Proteínas de Saccharomyces cerevisiae , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Transformada , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Femenino , Feto/citología , Proteínas Fúngicas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Locus Coeruleus/citología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuritas/fisiología , Neuronas/ultraestructura , Norepinefrina/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Embarazo , Regiones Promotoras Genéticas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiología , Proteína Elk-1 con Dominio etsRESUMEN
Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis-acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to beta-globin as a reporter was constructed. Transfection analysis revealed that cAMP-induced transcription of the secretogranin II promoter/beta-globin gene in septal and insulinoma cells. DNA-protein binding assays showed that recombinant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4-CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as cAMP-stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB-2, a negative-acting CRE-binding protein. These results indicate that competitive effects are likely to occur between CRE-bound transcriptional activators and repressors. We conclude that cAMP-stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell-type-specific manner, and is likely to depend on the balance between positive and negative CRE-binding proteins in a particular cell type.
Asunto(s)
AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas/genética , Transcripción Genética , 1-Metil-3-Isobutilxantina/farmacología , Animales , Secuencia de Bases , Bucladesina/farmacología , Diferenciación Celular/efectos de los fármacos , Cromograninas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Elementos de Facilitación Genéticos , Datos de Secuencia Molecular , Neuroblastoma/metabolismo , Células PC12 , Inhibidores de Fosfodiesterasa/farmacología , Unión Proteica , Biosíntesis de Proteínas , Ratas , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.
Asunto(s)
Citosina/análisis , Elementos de Facilitación Genéticos , Fructosa-Bifosfato Aldolasa/genética , Guanina/análisis , Proteínas Inmediatas-Precoces , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , Sistema Nervioso Central/enzimología , ADN , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Fructosa-Bifosfato Aldolasa/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Sistema Nervioso Periférico/enzimología , Unión Proteica , Ratas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
The distribution and biochemical features of the synapsin-like peptides recognized in Aplysia and Helix by various antibodies directed against mammalian synapsins were studied. The peptides can be extracted at low pH and are digested by collagenase; further, they can be phosphorylated by both protein kinase A and Ca2+/calmodulin-dependent protein kinase II. In the ganglia of both snails, they are associated with the soma of most neurons and with the neuropil; punctate immunostaining is present along the neurites. Using cocultures of a Helix serotoninergic neuron and of its target cell, we analysed the redistribution of the synapsin-like peptides during the formation of active synaptic contacts. When the presynaptic neuron is plated in isolation, both synapsin and serotonin immunoreactivities are restricted to the distal axonal segments and to the growth cones; in the presence of the target, the formation of a chemical connection is accompanied by redistribution of the synapsin and serotonin immunoreactivities that concentrate in highly fluorescent round spots scattered along the newly grown neurites located close to the target cell. Almost every spot that is stained for serotonin is also positive for synapsin. In the presynaptic cell plated alone, the number of these varicosity-like structures is substantially stable throughout the whole period; by contrast, when the presynaptic cell synapses the target, their number increases progressively parallel to the increase in the mean amplitude of cumulative excitatory postsynaptic potentials recorded at the same times. The data indicate that mollusc synapsin-like peptides to some extent resemble their mammalian homologues, although they are not exclusively localized in nerve terminals and their expression strongly correlates with the formation of active synaptic contacts.