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OBJECTIVE: (1) To define features and data items of a Patient Recruitment System (PRS); (2) to design a generic software architecture of such a system covering the requirements; (3) to identify implementation options available within different Hospital Information System (HIS) environments; (4) to implement five PRS following the architecture and utilizing the implementation options as proof of concept. METHODS: Existing PRS were reviewed and interviews with users and developers conducted. All reported PRS features were collected and prioritized according to their published success and user's request. Common feature sets were combined into software modules of a generic software architecture. Data items to process and transfer were identified for each of the modules. Each site collected implementation options available within their respective HIS environment for each module, provided a prototypical implementation based on available implementation possibilities and supported the patient recruitment of a clinical trial as a proof of concept. RESULTS: 24 commonly reported and requested features of a PRS were identified, 13 of them prioritized as being mandatory. A UML version 2 based software architecture containing 5 software modules covering these features was developed. 13 data item groups processed by the modules, thus required to be available electronically, have been identified. Several implementation options could be identified for each module, most of them being available at multiple sites. Utilizing available tools, a PRS could be implemented in each of the five participating German university hospitals. CONCLUSION: A set of required features and data items of a PRS has been described for the first time. The software architecture covers all features in a clear, well-defined way. The variety of implementation options and the prototypes show that it is possible to implement the given architecture in different HIS environments, thus enabling more sites to successfully support patient recruitment in clinical trials.
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Sistemas de Información en Hospital , Selección de Paciente , Programas Informáticos , Bases de Datos como Asunto , Alemania , Implementación de Plan de Salud , HumanosRESUMEN
BACKGROUND: Semantic interoperability between routine healthcare and clinical research is an unsolved issue, as information systems in the healthcare domain still use proprietary and site-specific data models. However, information exchange and data harmonization are essential for physicians and scientists if they want to collect and analyze data from different hospitals in order to build up registries and perform multicenter clinical trials. Consequently, there is a need for a standardized metadata exchange based on common data models. Currently this is mainly done by informatics experts instead of medical experts. OBJECTIVES: We propose to enable physicians to exchange, rate, comment and discuss their own medical data models in a collaborative web-based repository of medical forms in a standardized format. METHODS: Based on a comprehensive requirement analysis, a web-based portal for medical data models was specified. In this context, a data model is the technical specification (attributes, data types, value lists) of a medical form without any layout information. The CDISC Operational Data Model (ODM) was chosen as the appropriate format for the standardized representation of data models. The system was implemented with Ruby on Rails and applies web 2.0 technologies to provide a community based solution. Forms from different source systems - both routine care and clinical research - were converted into ODM format and uploaded into the portal. RESULTS: A portal for medical data models based on ODM-files was implemented (http://www.medical-data-models.org). Physicians are able to upload, comment, rate and download medical data models. More than 250 forms with approximately 8000 items are provided in different views (overview and detailed presentation) and in multiple languages. For instance, the portal contains forms from clinical and research information systems. CONCLUSION: The portal provides a system-independent repository for multilingual data models in ODM format which can be used by physicians. It serves as a platform for discussion and enables the exchange of multilingual medical data models in a standardized way.
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Persistently hyperphosphorylated Akt contributes to human oncogenesis and resistance to therapy. Triciribine (TCN) phosphate (TCN-P), the active metabolite of the Akt phosphorylation inhibitor TCN, is in clinical trials, but the mechanism by which TCN-P inhibits Akt phosphorylation is unknown. Here we show that in vitro, TCN-P inhibits neither Akt activity nor the phosphorylation of Akt S473 and T308 by mammalian target of rapamycin or phosphoinositide-dependent kinase 1. However, in intact cells, TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and phosphorylation of Akt. Surface plasmon resonance shows that TCN, but not TCN, binds Akt-derived pleckstrin homology (PH) domain (K(D): 690 nM). Furthermore, nuclear magnetic resonance spectroscopy shows that TCN-P, but not TCN, binds to the PH domain in the vicinity of the PIP3-binding pocket. Finally, constitutively active Akt mutants, Akt1-T308D/S473D and myr-Akt1, but not the transforming mutant Akt1-E17K, are resistant to TCN and rescue from its inhibition of proliferation and induction of apoptosis. Thus, the results of our studies indicate that TCN-P binds to the PH domain of Akt and blocks its recruitment to the membrane, and that the subsequent inhibition of Akt phosphorylation contributes to TCN-P antiproliferative and proapoptotic activities, suggesting that this drug may be beneficial to patients whose tumors express persistently phosphorylated Akt.
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Acenaftenos/metabolismo , Acenaftenos/farmacología , Membrana Celular/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ribonucleótidos/metabolismo , Ribonucleótidos/farmacología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Apoptosis , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Técnica del Anticuerpo Fluorescente , Amplificación de Genes , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Espectroscopía de Resonancia Magnética , Proteínas de la Membrana/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/química , Transducción de Señal , Resonancia por Plasmón de Superficie , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The interaction of the anti-cancer drug podophyllotoxin with a high-molecular-weight assembly of tubulin has been employed to produce three-dimensional crystals from avian erythrocyte tubulin as well as from pig brain tubulin. Avian erythrocyte tubulin crystals belong to the space group C2 with unit cell dimensions a = 740 A, b = 330 A, c = 460 A, beta = 128 degrees. The basis of these crystals is ring oligomers with a molecular mass of approximately 6 x 10(6) Da. So far, the crystals diffract to 8-A resolution and a first complete data set to 12-A resolution has been collected under cryogenic conditions. The crystals grew from conventionally purified tubulin consisting of multiple isoforms and different posttranslational modifications. Thus, the use of highly homogeneous tubulin preparations should improve the diffraction quality of these crystals.
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Antineoplásicos/química , Podofilotoxina/química , Tubulina (Proteína)/química , Animales , Aves , Cristalización , Eritrocitos/química , Microscopía Electrónica , Porcinos , Tubulina (Proteína)/ultraestructura , Difracción de Rayos XRESUMEN
We have performed a real time analysis of fluorescence-tagged vesicle and mitochondria movement in living CHO cells transfected with microtubule-associated protein tau or its microtubule-binding domain. tau does not alter the speed of moving vesicles, but it affects the frequencies of attachment and detachment to the microtubule tracks. Thus, tau decreases the run lengths both for plus-end and minus-end directed motion to an equal extent. Reversals from minus-end to plus-end directed movement of single vesicles are strongly reduced by tau, but reversals in the opposite direction (plus to minus) are not. Analogous effects are observed with the transport of mitochondria and even with that of vimentin intermediate filaments. The net effect is a directional bias in the minus-end direction of microtubules which leads to the retraction of mitochondria or vimentin IFs towards the cell center. The data suggest that tau can control intracellular trafficking by affecting the attachment and detachment cycle of the motors, in particular by reducing the attachment of kinesin to microtubules, whereas the movement itself is unaffected.
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Microtúbulos/fisiología , Proteínas Motoras Moleculares/fisiología , Proteínas tau/fisiología , Animales , Sitios de Unión , Transporte Biológico Activo , Células CHO , Cricetinae , Exocitosis , Colorantes Fluorescentes , Humanos , Filamentos Intermedios/fisiología , Cinesinas/fisiología , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/genética , Movimiento , Orgánulos/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Vimentina/fisiología , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein-driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins. Mitochondria fail to be transported to peripheral cell compartments and cluster in the vicinity of the microtubule-organizing center. The endoplasmic reticulum becomes less dense and no longer extends to the cell periphery. In differentiated N2a cells, the overexpression of tau leads to the disappearance of mitochondria from the neurites. These effects are caused by tau's binding to microtubules and slowing down intracellular transport by preferential impairment of plus-end-directed transport mediated by kinesin-like motor proteins. Since in Alzheimer's disease tau protein is elevated and mislocalized, these observations point to a possible cause for the gradual degeneration of neurons.
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Enfermedad de Alzheimer , Retículo Endoplásmico/metabolismo , Cinesinas/metabolismo , Mitocondrias/metabolismo , Taxoides , Proteínas tau/biosíntesis , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Animales , Transporte Biológico , Células CHO , Tamaño de la Célula , Centrosoma , Cricetinae , Docetaxel , Dineínas/metabolismo , Expresión Génica , Proteínas de Filamentos Intermediarios/metabolismo , Microinyecciones , Nocodazol/farmacología , Paclitaxel/análogos & derivados , Paclitaxel/farmacología , Transferrina/metabolismo , Células Tumorales Cultivadas , Proteínas tau/genéticaRESUMEN
In Alzheimer's disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80-90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau-microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer's disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.
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Enfermedad de Alzheimer/etiología , Ciclo Celular , Quinasas Ciclina-Dependientes , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Quinasa CDC2/metabolismo , Células CHO , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Cricetinae , Quinasa 5 Dependiente de la Ciclina , Expresión Génica , Glucógeno Sintasa Quinasa 3 , Células HeLa , Humanos , Interfase , Microtúbulos/metabolismo , Mitosis , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Serina/metabolismo , Proteínas tau/genéticaRESUMEN
The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. We have shown recently that a novel protein kinase, termed p110 microtubule-affinity regulating kinase ("MARK"), phosphorylates microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and causes the detachment of tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H.E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Here we show that p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells.
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Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Fosforilación , Unión Proteica , RatasRESUMEN
This paper summarizes recent structural and functional studies on tau protein, its interactions with microtubules, its self-assembly into paired helical filaments (PHF)-like fibers, and its modification by phosphorylation. The structure of tau in solution resembles that of a random coil. Both tau and Alzheimer PHFs have very little secondary structure, making it improbable that the assembly of tau into PHFs is based on interacting beta sheets. Tau's binding to microtubules can be described by a "jaws" effect. The domain containing the repeats binds very weakly, while the flanking regions (jaws) bind strongly, even without the repeats. However, only the combination of flanking regions and repeats makes binding productive in terms of microtubule nucleation and assembly. Although the majority of Alzheimer-like phosphorylation sites are outside the repeats they have only a weak influence on binding, whereas the phosphorylation at Ser262 inside the repeats inhibits binding and makes microtubules dynamically unstable. This site can be phosphorylated by kinases present in brain tissue, and it is uniquely phosphorylated in Alzheimer brain.
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Proteínas tau/química , Proteínas tau/fisiología , Microtúbulos/fisiología , Conformación Molecular , Fosforilación , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.
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Quinasas Ciclina-Dependientes , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Quinasa 5 Dependiente de la Ciclina , Variación Genética , Sustancias Macromoleculares , Microscopía por Video , Datos de Secuencia Molecular , Neuronas/enzimología , Fosforilación , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
We consider the interactions of tau protein with microtubules from two points of view, phosphorylation and domain structure. Tau can be phosphorylated at many sites and by several kinases, notably by proline-directed kinases (MAPK, GSK-3, cdk5) which generate Alzheimer-like antibody epitopes. Other kinases phosphorylate Ser 262, a site that has a particularly pronounced influence on the affinity of tau for microtubules. All of these sites can be cleared by phosphatases PP-2a and calcineurin. The site Ser262 lies within the repeat domain of tau. However, when probing the domains of tau for their effects on microtubule binding, nucleation, assembly, or bundling, the repeat domain has only a weak influence. Whereas the repeat domain of tau binds to microtubules with low affinity, repeat-less tau binds strongly yet unproductively in terms of microtubule assembly. Productive binding of tau to microtubules depends on the combination of (some) repeats with the flanking regions, as if the flanking regions acted as "jaws" for the proper positioning of tau on the microtubule surface.
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Microtúbulos/química , Proteínas tau/química , Animales , Humanos , Microtúbulos/metabolismo , Fosforilación , Unión Proteica , Proteínas tau/metabolismoRESUMEN
Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E. (1993) Neuron 11, 153-163). Here we describe the purification and characterization of a protein-serine kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtuble-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.
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Enfermedad de Alzheimer/metabolismo , Encéfalo/enzimología , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Serina , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía por Intercambio Iónico , Clonación Molecular , Escherichia coli , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Fosforilación , Fosfoserina/análisis , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Porcinos , Tripsina , Tubulina (Proteína)/aislamiento & purificación , Proteínas tau/biosíntesis , Proteínas tau/aislamiento & purificaciónRESUMEN
The role of the neuronal microtubule-associated protein tau has been studied by generating a series of tau constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N- or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N-terminal inserts. The interaction of the mutant tau proteins with microtubules was judged by several independent methods. (i) Direct binding assays between tau and taxol-stabilized microtubules yield dissociation constants and stoichiometries. (ii) Light scattering and X-ray scattering of assembling microtubule solutions reflect the capacity of tau to promote microtubule nucleation, elongation, and bundling in bulk solution. (iii) Dark field microscopy of assembling microtubules allows one to assess the efficiency of nucleation and bundling separately. The repeat region alone, the N-terminal domains alone, or the C-terminal tail alone binds only weakly to microtubules. However, binding is strongly enhanced by combinations such as the repeat region plus one or both of the flanking regions which could be viewed as "jaws" for tau on the microtubule surface (the proline-rich domain P upstream of the repeats and the "fifth" repeat R' downstream). Such combinations make tau's binding productive in terms of microtubule assembly and stabilization, while the combination of the flanking regions without repeats binds only unproductively. Efficient nucleation parallels strong binding in most cases, i.e., when a construct binds tightly to microtubules, it also nucleates them efficiently and vice versa. In addition, the proline-rich domain P in combination with the repeats R or the flanking domain R' causes pronounced bundling. This effect disappears when the N-terminal domains (acidic or basic) are added on, suggesting that the tau isoforms are not "bundling proteins" in the proper sense. In spite of the wide range of binding strength and nucleation efficiency, the stoichiometries of binding are rather reproducible (around 0.5 tau/tubulin dimer); this is in remarkable contrast to the effect of certain types of phosphorylation which can strongly reduce the stoichiometry.
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Microtúbulos/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Humanos , Microscopía/métodos , Microtúbulos/ultraestructura , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Dispersión de Radiación , Relación Estructura-Actividad , Grabación en Video , Rayos XRESUMEN
Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.
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Eritrocitos/ultraestructura , Microtúbulos/ultraestructura , Animales , Encéfalo/metabolismo , Encéfalo/ultraestructura , Pollos , Eritrocitos/metabolismo , Técnicas In Vitro , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Biológicos , Fosfatos/farmacología , Dispersión de Radiación , Especificidad de la Especie , Porcinos , Termodinámica , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestructuraRESUMEN
Catalytic (C) and regulatory subunits (RI, RII) of cAMP-dependent protein kinase (PKA) were localized in cell nuclei of various rat tissues by immunogold electron microscopy. A specific labeling within the interchromatin space was found to be associated with perichromatin fibrils, interchromatin granules, and coiled bodies. Condensed chromatin and perichromatin granules were not labeled. In the nucleolus, most gold particles were found in the dense fibrillar component. Quantitation of labeling densities for the C-subunit from cells at different stages of spermatogenesis indicated that the nuclear concentration of PKA varies inversely with the amount of condensed chromatin. No parallel alterations in C-immunoreactivities were observed in the cytoplasm. Immunofluorescence studies on polytene chromosomes of Chironomus thummi with a confocal laser scanning microscope showed a significantly more intense labeling of the C-subunit in transcriptionally active sites (puffs) as compared to inactive bands. Our results indicate that local concentrations of PKA in the nucleus have physiological significance in the control of gene activity. In addition, they suggest that PKA plays a regulatory role also in RNA processing.
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Nucléolo Celular/enzimología , Cromatina/química , Proteínas Quinasas/análisis , Transcripción Genética , Animales , Nucléolo Celular/ultraestructura , Chironomidae , Cromatina/ultraestructura , Inmunohistoquímica , Masculino , Ratas , Ratas Wistar , Glándulas Salivales/enzimología , Glándulas Salivales/ultraestructura , Espermatogénesis , Espermatozoides/enzimología , Espermatozoides/ultraestructuraRESUMEN
The catalytic subunit of cAMP-dependent protein kinases was localized in microtubules and neurofilaments by immunogold electron microscopy. In microtubules, the label was similarly distributed as an immunolabel for the microtubule associated protein MAP 2. The neurofilaments showed no reaction with the MAP 2-antiserum. Our results support the suggestion of an in vivo role of cAMP-dependent protein kinases in the regulation of microtubules. In addition, this is the first demonstration that cAMP-dependent protein kinase is associated with neurofilaments.
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Encéfalo/enzimología , Filamentos Intermedios/enzimología , Microtúbulos/enzimología , Proteínas Quinasas/metabolismo , Animales , Encéfalo/ultraestructura , Inmunohistoquímica , Masculino , Ratas , Ratas EndogámicasRESUMEN
Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.