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1.
Scand J Med Sci Sports ; 21(6): e64-71, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20561270

RESUMEN

Despite the importance of recuperation, few have studied the impact of rest periods on injury prevention. We determined the effect of rest days (breaks) on injury rates and treatments using electronic injury records from an acrobatic circus company that employs former world-class athletes as acrobats. To account for accumulated fatigue, we considered breaks across SD3 (third consecutive week of 1-day rest) to SD6 as a single exposure level (SD3-6), and vacation and DD (2-day rest) as a single exposure level. Medical attention injury rates were increased post- vs pre-break {rate ratio 1.45 [95% confidence intervals (95% CI): 1.22-1.73]} with less of an effect for 1-day time loss [1.25 (95% CI: 0.58-2.67)] and 15-day time loss [1.10 (95% CI: 0.26-4.56)]. However, the increase in injury rate post break for SD3-6 was similar to that of DD-Vacation (P=0.48, 0.53, and 0.65) for medical attention, and both ≥1 day and ≥15 days time loss, respectively. The increase in the number of treatments post-break was less for SD3-6 vs DD-vacation. Our findings suggest that 2-day breaks every four to 6 weeks may be sufficient to avoid an increasing injury rate due to cumulative fatigue in professional acrobatic circus artists.


Asunto(s)
Traumatismos en Atletas/prevención & control , Músculo Esquelético/lesiones , Descanso/fisiología , Adulto , Estudios de Cohortes , Femenino , Humanos , Masculino , Fatiga Muscular/fisiología , Recuperación de la Función/fisiología , Factores de Riesgo , Adulto Joven
2.
J Cell Biol ; 153(1): 177-90, 2001 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-11285284

RESUMEN

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the approximately 300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.


Asunto(s)
Células Cromafines/fisiología , Vesículas Secretoras/fisiología , Actinas/metabolismo , Animales , Factor Natriurético Atrial/metabolismo , Toxinas Botulínicas Tipo A , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Bovinos , Membrana Celular/fisiología , Citoesqueleto/metabolismo , Difusión , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Microscopía Fluorescente/métodos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas R-SNARE , Proteínas Recombinantes de Fusión/metabolismo , Proteína 25 Asociada a Sinaptosomas , Toxina Tetánica , Tiazoles/metabolismo , Tiazolidinas , Factores de Tiempo
3.
Science ; 242(4878): 577-9, 1988 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-2845583

RESUMEN

The alpha and beta subunits of the gamma-aminobutyric acidA (GABAA) receptor were expressed individually in Xenopus oocytes by injection of RNA synthesized from their cloned DNAs. GABA-sensitive chloride channels were detected several days after injection with any one of three different alpha RNAs (alpha 1, alpha 2, and alpha 3) or with beta RNA. The channels induced by each of the alpha-subunit RNAs were indistinguishable, they had multiple conductance levels (10, 19, 28, and 42 picosiemens), and their activity was potentiated by pentobarbital and inhibited by picrotoxin. The beta channels usually expressed poorly but showed similar single channel conductance levels (10, 18, 27, and 40 picosiemens), potentiation by pentobarbital and inhibition by picrotoxin. The finding that both alpha and beta subunits, examined separately, form GABA-sensitive ion channels with permeation properties and regulatory sites characteristic of the native receptor suggests that the amino acid sequences that confer these properties are within the homologous domains shared by the subunits.


Asunto(s)
Cloruros/fisiología , Proteínas de la Membrana/fisiología , Receptores de GABA-A/fisiología , Animales , Canales de Cloruro , Clonación Molecular , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Sustancias Macromoleculares , Picrotoxina/farmacología , ARN Mensajero/administración & dosificación , Relación Estructura-Actividad , Xenopus laevis , Ácido gamma-Aminobutírico/farmacología
4.
Neuron ; 6(4): 557-63, 1991 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-1849723

RESUMEN

Many neurotransmitters and hormones regulate secretion from endocrine cells and neurons by modulating voltage-gated Ca2+ channels. One proposed mechanism of neurotransmitter inhibition involves protein kinase C, activated by diacylglycerol, a product of phosphatidyl-inositol inositol hydrolysis. Here we show that thyrotropin-releasing hormone (TRH), a neuropeptide that modulates hormone secretion from pituitary tumor cells, inhibits Ca2+ channels via the other limb of the phosphatidylinositol signaling system: TRH causes inositol trisphosphate-triggered Ca2+ release from intracellular organelles, thus causing Ca2(+)-dependent inactivation of Ca2+ channels. Elevation of intracellular Ca2+ concentration is coincident with the onset of TRH-induced inhibition and is necessary and sufficient for its occurrence. The inhibition is blocked by introducing Ca2+ buffers into cells and mimicked by a variety of agents that mobilize Ca2+. Treatments that suppress protein kinase C have no effect on the inhibition. Hence inactivation of Ca2+ channels occurs not only as a result of Ca2+ influx through plasma membrane channels, but also via neurotransmitter-induced Ca2+ mobilization. This phenomenon may be common but overlooked because of the routine use of Ca2+ buffers in patch-clamp electrodes.


Asunto(s)
Canales de Calcio/efectos de los fármacos , Calcio/metabolismo , Membranas Intracelulares/metabolismo , Activación del Canal Iónico/fisiología , Neuropéptidos/farmacología , Electrofisiología , Proteína Quinasa C/fisiología , Hormona Liberadora de Tirotropina/farmacología
5.
Neuron ; 1(9): 773-81, 1988 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-2856103

RESUMEN

Biochemical and immunological studies indicate that the GABAA receptor contains at least two types of subunit. Here we report that coexpression of two GABAA receptor subunit clones (alpha and beta) in Xenopus oocytes yields receptors with many biophysical properties of native GABAA receptors. These include ion selectivity, multiple single-channel conductance states, voltage-dependent gating and rectification, and complex desensitization kinetics. Furthermore, the receptors are competitively inhibited by bicuculline and display the expected allosteric and agonist effects of the barbiturate pentobarbital. The expressed receptors, however, appear to be activated by one molecule of GABA instead of two and fail to show potentiation by benzodiazepines. This implies that an additional factor(s) or subunit(s) is required for the reconstitution of a fully functional GABAA receptor.


Asunto(s)
Bicuculina/farmacología , Oocitos/fisiología , Pentobarbital/farmacología , Receptores de GABA-A/fisiología , Xenopus laevis/fisiología , Animales , Femenino , Potenciales de la Membrana/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Xenopus laevis/metabolismo
6.
Neuron ; 11(2): 359-69, 1993 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8352944

RESUMEN

Glucocorticoids specifically increase Kv1.5 K+ channel mRNA in normal and clonal (GH3) rat pituitary cells. Here, we demonstrate that dexamethasone, a glucocorticoid agonist, rapidly induces Kv1.5 gene transcription, but does not affect Kv1.5 mRNA turnover (t1/2 approximately 0.5 hr) in GH3 cells. Immunoblots indicate that the steroid also increases the expression of the 76 kd Kv1.5 protein approximately 3-fold within 12 hr without altering its half-life (t1/2 approximately 4 hr). In contrast, Kv1.4 protein expression is unaffected. Finally, we find that the induction of Kv1.5 protein is associated with an increase in a noninactivating component of the voltage-gated K+ current. Our results indicate that hormones and neurotransmitters may act within hours to regulate excitability by controlling K+ channel gene expression.


Asunto(s)
Dexametasona/farmacología , Hipófisis/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Células Clonales , Hipófisis/citología , ARN Mensajero/metabolismo
7.
Neuron ; 19(5): 1095-102, 1997 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9390522

RESUMEN

Neuropeptides are slowly released from a limited pool of secretory granules. To visualize this process, GFP-tagged preproatrial natriuretic factor (ANF) was expressed in nerve growth factor-treated PC12 cells. Biochemical and microfluorimetric experiments demonstrate that proANF-EGFP is packaged in granules that accumulate at neurite endings and is released in a Ca2+-dependent manner by secretagogs. Confocal microscopy shows that secretion is associated with depletion of granules distributed throughout the terminal. Fluorescence recovery after photobleaching and time-lapse particle tracking reveal that only a subpopulation of cytoplasmic secretory granules, similar in size to the releasable pool, can move quickly enough (D = 6 x 10(-11) cm2/s) to support release. Therefore, sustained secretory responses are limited by the number of mobile granules and their slow rate of diffusion.


Asunto(s)
Gránulos Citoplasmáticos/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Animales , Factor Natriurético Atrial/metabolismo , Difusión , Fluorescencia , Variación Genética , Proteínas Fluorescentes Verdes , Cinética , Proteínas Luminiscentes/genética , Células PC12 , Precursores de Proteínas/metabolismo , Ratas , Lugares Marcados de Secuencia , Estimulación Química , Distribución Tisular
8.
Int J Obes (Lond) ; 32(2): 201-10, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18278059

RESUMEN

OBJECTIVE: Perform a systematic review of studies reporting on the association between maternal prenatal cigarette smoking and child overweight. DESIGN: Meta-analysis of observational studies. DATA SOURCES: Medline search and review of reference lists among studies published through June 2006. REVIEW METHODS: Included studies reported an association between maternal smoking during pregnancy and risk of overweight among children at least 2 years of age. We did not include in the meta-analysis studies that provided only a continuous measure of adiposity, although those studies are discussed separately. RESULTS: Based on results of 84 563 children reported in 14 observational studies, children whose mothers smoked during pregnancy were at elevated risk for overweight (pooled adjusted odds ratio (OR) 1.50, 95% CI: 1.36, 1.65) at ages 3-33 years, compared with children whose mothers did not smoke during pregnancy. The pooled estimate from unadjusted odds ratios (OR 1.52, 95% CI: 1.36, 1.69) was similar to the adjusted estimate, suggesting that sociodemographic and behavioral differences between smokers and nonsmokers did not explain the observed association. Although we observed evidence for publication bias, simulating a symmetric set of studies yielded a similar estimate (OR 1.40, 95% CI: 1.26, 1.55). CONCLUSIONS: Prenatal smoking exposure appears to increase rates of overweight in childhood. In parts of the world undergoing the epidemiologic transition, the continuing increase in smoking among young women could contribute to spiraling increases in rates of obesity-related health outcomes in the 21st century.


Asunto(s)
Sobrepeso/embriología , Efectos Tardíos de la Exposición Prenatal , Fumar/efectos adversos , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Exposición Materna , Oportunidad Relativa , Sobrepeso/epidemiología , Embarazo , Factores de Riesgo
9.
Cell Death Differ ; 13(4): 661-7, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16273079

RESUMEN

Apoptosis in cortical neurons requires efflux of cytoplasmic potassium mediated by a surge in Kv2.1 channel activity. Pharmacological blockade or molecular disruption of these channels in neurons prevents apoptotic cell death, while ectopic expression of Kv2.1 channels promotes apoptosis in non-neuronal cells. Here, we use a cysteine-containing mutant of Kv2.1 and a thiol-reactive covalent inhibitor to demonstrate that the increase in K+ current during apoptosis is due to de novo insertion of functional channels into the plasma membrane. Biotinylation experiments confirmed the delivery of additional Kv2.1 protein to the cell surface following an apoptotic stimulus. Finally, expression of botulinum neurotoxins that cleave syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) blocked upregulation of surface Kv2.1 channels in cortical neurons, suggesting that target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins support proapoptotic delivery of K+ channels. These data indicate that trafficking of Kv2.1 channels to the plasma membrane causes the apoptotic surge in K+ current.


Asunto(s)
Apoptosis , Membrana Celular/metabolismo , Neuronas/metabolismo , Canales de Potasio Shab/metabolismo , Animales , Biotinilación , Células CHO , Membrana Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/embriología , Cricetinae , Cricetulus , Potenciales de la Membrana , Neuronas/efectos de los fármacos , Neuronas/patología , Potasio/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas SNARE/metabolismo , Canales de Potasio Shab/biosíntesis , Canales de Potasio Shab/genética , Transfección
10.
Neuroscience ; 143(1): 1-6, 2006 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-17027171

RESUMEN

6-Hydroxydopamine (6-OHDA), a neurotoxic substrate of the dopamine transporter (DAT), is widely used in Parkinson's disease models. However, the molecular mechanisms underlying 6-OHDA's selectivity for dopamine neurons and the injurious sequelae that it triggers are not well understood. We tested whether ectopic expression of DAT induces sensitivity to 6-OHDA in non-dopaminergic rat cortical neurons and evaluated the contribution of voltage-dependent potassium channel (Kv)-dependent apoptosis to the toxicity of this compound in rat cortical and midbrain dopamine neurons. Cortical neurons expressing DAT accumulated dopamine and were highly vulnerable to 6-OHDA. Pharmacological inhibition of DAT completely blocked this toxicity. We also observed a p38-dependent Kv current surge in DAT-expressing cortical neurons exposed to 6-OHDA, and p38 antagonists and Kv channel blockers were neuroprotective in this model. Thus, DAT-mediated uptake of 6-OHDA recruited the oxidant-induced Kv channel dependent cell death pathway present in cortical neurons. Finally, we report that 6-OHDA also increased Kv currents in cultured midbrain dopamine neurons and this toxicity was blocked with Kv channel antagonists. We conclude that native DAT expression accounts for the dopamine neuron specific toxicity of 6-OHDA. Following uptake, 6-OHDA triggers the oxidant-associated Kv channel-dependent cell death pathway that is conserved in non-dopaminergic cortical neurons and midbrain dopamine neurons.


Asunto(s)
Adrenérgicos/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Neuronas/efectos de los fármacos , Oxidopamina/farmacología , Canales de Potasio con Entrada de Voltaje/fisiología , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Tetraetilamonio/farmacología , Transfección/métodos
11.
Circ Res ; 89(3): E23-9, 2001 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-11485981

RESUMEN

How vesicles are born in the trans-Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 microm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles (r=0.71, P<0.0005). The deletion of the acidic N-terminal proANP[1-44] or point mutations (glu(23,24)-->gln(23,24)) change size and shape-but not velocity-of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the trans-Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate-docking or not docking-at the plasma membrane. The full text of this article is available at http://www.circresaha.org.


Asunto(s)
Atrios Cardíacos/metabolismo , Miocardio/metabolismo , Vesículas Secretoras/metabolismo , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/genética , Sitios de Unión/fisiología , Transporte Biológico/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Proteínas Fluorescentes Verdes , Atrios Cardíacos/ultraestructura , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Proteínas Luminiscentes/genética , Ratones , Microscopía Inmunoelectrónica , Microesferas , Mutagénesis Sitio-Dirigida , Miocardio/ultraestructura , Tamaño de la Partícula , Precursores de Proteínas/genética , Señales de Clasificación de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Vesículas Secretoras/ultraestructura , Transducción de Señal/fisiología , Relación Estructura-Actividad , Red trans-Golgi/metabolismo
12.
Circ Res ; 88(5): 476-82, 2001 Mar 16.
Artículo en Inglés | MEDLINE | ID: mdl-11249870

RESUMEN

Hypertrophied cardiac myocytes exhibit prolonged action potentials and decreased transient outward potassium current (I(to)). Because Kv4.3 is a major contributor to I:(to), we studied regulation of its expression in neonatal rat cardiac myocytes in response to the known stimulators of cardiac myocyte hypertrophy, angiotensin II (Ang II) and phenylephrine (PE). RNase protection assays and immunoblots revealed that Ang II and PE each downregulate Kv4.3 mRNA and protein. However, although PE induces a faster and more extensive hypertrophic response than Ang II, the PE effect on Kv4.3 mRNA develops slowly and is sustained, whereas Ang II rapidly and transiently decreases Kv4.3 mRNA expression. Turnover measurements revealed that Kv4.3 mRNA is very stable, with a half-life >20 hours. This suggests that Ang II must destabilize the channel mRNA. In contrast, PE does not affect the rate of Kv4.3 mRNA degradation. To test for transcriptional regulation, the 5' flanking region of the rat Kv4.3 gene was cloned, and Kv4.3 promoter-reporter constructs were expressed in cardiac myocytes. Whereas Ang II was found to have no effect on transcription, PE inhibits Kv4.3 promoter activity. Pharmacological experiments also indicate that PE and Ang II act independently to downregulate Kv4.3 gene expression. Thus, regulation of Kv4.3 gene expression is not a simple secondary response to hypertrophy. Rather, Ang II and PE use different mechanisms to decrease Kv4.3 channel expression in neonatal rat cardiac myocytes.


Asunto(s)
Angiotensina II/farmacología , Miocardio/metabolismo , Fenilefrina/farmacología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/efectos de los fármacos , Antagonistas de Receptores de Angiotensina , Animales , Animales Recién Nacidos , Células Cultivadas , ADN/genética , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Miocardio/citología , Canales de Potasio/genética , Canales de Potasio/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales de Potasio Shal , Tetrazoles/farmacología , Factores de Tiempo , Transcripción Genética/efectos de los fármacos
13.
Oncogene ; 18(48): 6621-34, 1999 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-10597267

RESUMEN

Mad proteins are basic-helix-loop-helix-leucine zipper (bHLH-ZIP)-containing members of the myc oncoprotein network. They interact with the bHLH-ZIP protein max, compete for the same DNA binding sites as myc-max heterodimers and down-regulate myc-responsive genes. Using the bHLH-ZIP domain of mad1 as a yeast two-hybrid 'bait', we identified Mmip-2, a novel RING finger protein that interacts with all mad members, but weakly or not at all with c-myc, max or unrelated bHLH or bZIP proteins. The mad1-Mmip-2 interaction is mediated by the ZIP domain in the former protein and by at least two regions in the latter which do not include the RING finger. Mmip-2 can disrupt max-mad DNA binding and can reverse the suppressive effects of mad proteins on c-myc-responsive target genes and on c-myc + ras-mediated focus formation in fibroblasts. Tagging with spectral variants of green fluorescent protein showed that Mmip-2 and mad proteins reside in separate cytoplasmic and nuclear compartments, respectively. When co-expressed, however, the proteins interact and translocate to the cellular compartment occupied by the more abundant protein. These observations suggest a novel way by which Mmip-2 can modulate the transcriptional activity of myc oncoproteins.


Asunto(s)
Proteínas Portadoras , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras , Factores de Transcripción/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular , Clonación Molecular , ADN Complementario , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos
14.
Oncogene ; 20(34): 4650-64, 2001 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-11498788

RESUMEN

Members of the Myc oncoprotein network (c-Myc, Max, and Mad) play important roles in proliferation, differentiation, and apoptosis. We expressed chimeric green fluorescent protein (GFP) fusions of c-Myc, Max, and three Mad proteins in fibroblasts. Individually, c-Myc and Mad proteins localized in subnuclear speckles, whereas Max assumed a homogeneous nuclear pattern. These distributions were co-dominant and dynamic, however, as each protein assumed the pattern of its heterodimeric partner when the latter was co-expressed at a higher level. Deletion mapping of two Mad members, Mad1 and Mxi1, demonstrated that the domains responsible for nuclear localization and speckling are separable. A non-speckling Mxi1 mutant was also less effective as a transcriptional repressor than wild-type Mxi1. c-Myc nuclear speckles were distinct from SC-35 domains involved in mRNA processing. However, in the presence of co-expressed Max, c-Myc, but not Mad, co-localized to a subset of SC-35 loci. These results show that Myc network proteins comprise dynamic subnuclear structures and behave co-dominantly when co-expressed with their normal heterodimerization partners. In addition, c-Myc-Max heterodimers, but not Max-Mad heterodimers, localize to foci actively engaged in pre-mRNA transcription/processing. These findings suggest novel means by which Myc network members promote transcriptional activation or repression.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Western Blotting , Células COS , Compartimento Celular , Proteínas de Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Inhibidor NF-kappaB alfa , Proteínas Nucleares , Fosfoproteínas/química , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/genética , Procesamiento Postranscripcional del ARN , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Proteínas Supresoras de Tumor
15.
Biochim Biophys Acta ; 1265(1): 22-8, 1995 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-7857980

RESUMEN

The role of protein kinases in maintaining basal expression of voltage-gated K+ channel mRNA was examined in GH3 clonal pituitary cells. Nonspecific inhibition of protein kinases with H7 or staurosporine markedly decreases Kv1.5 K+ channel gene transcription and mRNA without producing a substantial change in Kv1.4 mRNA. Selective inhibitors for protein kinase C, Ca(2+)-calmodulin kinases, and tyrosine kinases do not affect Kv1.5 mRNA expression. In contrast, the Rp-diastereomer of adenosine 3',5'-cyclic monophosphorothioate, a specific inhibitor of protein kinase A, partially inhibits Kv1.5 mRNA expression (approximately 40%), and this effect was antagonized by 8-bromo-adenosine 3',5'-cyclic monophosphate. Thus, protein kinase A and at least one other kinase are required for basal Kv1.5 mRNA expression in pituitary cells.


Asunto(s)
Hipófisis/metabolismo , Canales de Potasio/genética , Inhibidores de Proteínas Quinasas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Alcaloides/farmacología , Animales , Células Clonales , Expresión Génica , Isoquinolinas/farmacología , Piperazinas/farmacología , ARN Mensajero/metabolismo , Ratas , Estaurosporina , Transcripción Genética/efectos de los fármacos
16.
Biochim Biophys Acta ; 813(2): 213-20, 1985 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-3970922

RESUMEN

Potassium accumulation associated with outward membrane potassium current was investigated experimentally in Myxicola giant axon. During prolonged voltage-clamp pulses to positive transmembrane potentials, the K+ equilibrium potential may approach zero mV, suggesting massive K+ accumulation outside the axonal membrane to concentrations many-fold higher than those in the bathing medium. The potassium accumulation can be satisfactorily described by a three-compartment model, consisting of the nerve fiber, a restricted physiological periaxonal space and the bulk solution. The average thickness, theta, of the periaxonal space is calculated as 177 +/- 59 A, i.e., comparable to that in the squid, while the permeability coefficient of the external barrier, PKs, was calculated to be (1.4 +/- 0.4) X 10(-4) cm/s. These conclusions are well supported by morphological study.


Asunto(s)
Axones/fisiología , Poliquetos/metabolismo , Potasio/metabolismo , Animales , Axones/ultraestructura , Cationes Monovalentes , Decapodiformes , Conductividad Eléctrica , Electrofisiología , Potenciales de la Membrana , Microscopía Electrónica , Modelos Biológicos , Permeabilidad
17.
J Gen Physiol ; 108(1): 1-11, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8817380

RESUMEN

Whole-cell L-type Ca2+ channel current was recorded in GH3 clonal rat pituitary cells using Ba2+ as a charge carrier. In the presence of the dihydropyridine agonist Bay K 8644, deactivation was best described by two exponential components with time constants of approximately 2 and approximately 8 ms when recorded at -40 mV. The slow component activated at more negative potentials than the fast component: Half-maximal activation for the slow and fast components occurred at approximately -15 and approximately 1 mV, respectively. The fast component was more sensitive to enhancement by racemic Bay K 8644 than the slow component: ED50fast = approximately 21 nM, ED50slow = approximately 74 nM. Thyrotropin-releasing hormone (TRH; 1 microM) inhibited the slow component by approximately 46%, whereas the fast component was inhibited by approximately 22%. TRH inhibition of total L-current showed some voltage dependence, but each Bay K 8644-revealed component of L-current was inhibited in a voltage-independent manner. Therefore, the apparent voltage dependence of TRH action is derived from complexities in channel gating rather than from relief of inhibition at high voltages. In summary, Bay K 8644-enhanced L-currents in GH3 cells consist of two components with different sensitivities to voltage, racemic Bay K 8644, and the neuropeptide TRH.


Asunto(s)
Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Canales de Calcio/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Hipófisis/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Técnicas de Placa-Clamp , Ratas
18.
J Gen Physiol ; 108(1): 13-26, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8817381

RESUMEN

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell-attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.


Asunto(s)
Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Canales de Calcio/fisiología , Potenciales de la Membrana/fisiología , Hipófisis/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Células Cultivadas , Hipófisis/efectos de los fármacos , Ratas
19.
J Gen Physiol ; 113(1): 71-80, 1999 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9874689

RESUMEN

PDZ-domain-containing proteins such as PSD-95 have been implicated in the targeting and clustering of membrane proteins. Biochemical and immunohistochemical studies indicate that PSD-95 recognizes COOH-terminal S/TXV sequences present in Kv1 K+ channels. However, the effect of binding a PDZ domain on a target protein has not been studied in live cells. In the present study, a green fluorescent protein-Kv1.4 fusion protein is used to study the effect of PSD-95 on channel movement. Fluorescence recovery after photobleaching showed that PSD-95 can immobilize K+ channels in the plasma membrane in an all-or-none manner. Furthermore, time lapse imaging showed that channel clusters formed in the presence of PSD-95 are stable in size, shape, and position. As expected from previous reports, two green fluorescent protein-tagged COOH-terminal variants of Kv1.4, Delta15 and V655A, are not clustered by PSD-95. However, coexpression of PSD-95 with V655A, but not Delta15, leads to the appearance of PSD-95 immunoreactivity in the plasma membrane. Furthermore, fluorescence recovery after photobleaching studies show that V655A channels are immobilized by PSD-95. Thus, V655A channels can interact with PSD-95 in a manner that leads to channel immobilization, but not clustering. These experiments document for the first time that PSD-95 immobilizes target proteins. Additionally, the data presented here demonstrate that the structural requirements for protein clustering and immobilization by PSD-95 are distinct.


Asunto(s)
Proteínas del Tejido Nervioso/química , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/química , Animales , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Clonación Molecular , Homólogo 4 de la Proteína Discs Large , Proteínas Fluorescentes Verdes , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular , Canal de Potasio Kv1.4 , Proteínas Luminiscentes/química , Proteínas de la Membrana/química , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Conformación Proteica , Ratas
20.
Mol Endocrinol ; 5(12): 1903-8, 1991 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-1791837

RESUMEN

Glucocorticoid hormones are released as part of the stress response and regulate secretion by the pituitary. Since the activity of ion channels also influences secretion, we examined the effect of the glucocorticoid agonist dexamethasone on ion channel expression. K+ channel mRNA was detected in rat hypothalamus and anterior pituitary, with probes derived from the rat Kv1 gene, a member of the mammalian voltage-gated K+ channel superfamily. High levels were also detected in PRL-secreting clonal (GH3 and GH4C1) rat pituitary cells. Dexamethasone rapidly increased the steady state concentration of Kv1 mRNA in GH3 cells in a dose-dependent manner. This change in gene expression was accompanied by an increase in whole cell voltage-gated K+ current [lk(i)] with similar pharmacology to the Kv1 gene product. Our findings indicate that hormones may act directly on excitable cells to produce long term effects on electrical activity and secretion by regulating K+ channel expression.


Asunto(s)
Dexametasona/farmacología , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Canales de Potasio/genética , ARN Mensajero/análisis , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/patología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Adenohipófisis/efectos de los fármacos , Adenohipófisis/metabolismo , Adenohipófisis/patología , Neoplasias Hipofisarias/metabolismo , Potasio/farmacocinética , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Prolactina/metabolismo , ARN Mensajero/genética , ARN Mensajero/fisiología , Ratas , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología
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