RESUMEN
In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.
Asunto(s)
División Celular , Cromosomas/fisiología , Aneuploidia , Animales , Segregación Cromosómica , Humanos , Cinetocoros , Mitosis , Neoplasias/genética , Saccharomyces cerevisiae/genética , Huso AcromáticoRESUMEN
The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described (Freeman et al., 1992). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Ciclina B/metabolismo , Oocitos/metabolismo , Proteínas Proto-Oncogénicas c-mos/metabolismo , Xenopus/metabolismo , Animales , Western Blotting , Estabilidad de Enzimas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Mapeo Peptídico , Fosforilación , Pruebas de Precipitina , Proteínas Recombinantes/metabolismo , Ubiquitina/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
Members of the polo-like family of protein kinases have been involved in the control of APC (anaphase-promoting complex) during the cell cycle, yet how they activate APC is not understood in any detail. In Xenopus oocytes, Ca2+-dependent degradation of cyclin B associated with release from arrest at second meiotic metaphase was demonstrated to require the polo-like kinase Plx1. The aim of the present study was to examine, beyond Ca2+-dependent resumption of meiosis, the possible role of Plx1 in the control of cyclin degradation during the early mitotic cell cycle. Plx1 was found to be dispensable for MPF to turn on the cyclin degradation machinery. However, it is required to prevent premature inactivation of the APC-dependent proteolytic pathway. Microcystin suppresses the requirement for Plx1 in both Ca2+-dependent exit from meiosis, associated with degradation of both cyclin B and A downstream of CaMK2 activation, and prevention of premature APC(Fizzy) inactivation in the early mitotic cell cycle. These results are consistent with the view that Plx1 antagonizes an unidentified microcystin-sensitive phosphatase that inactivates APC(Fizzy).
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ligasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Complejos de Ubiquitina-Proteína Ligasa , Proteínas de Xenopus , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteína Quinasa CDC2/metabolismo , Proteínas Cdc20 , Ciclo Celular , Ciclina B/metabolismo , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Microcistinas , Complejos Multienzimáticos/metabolismo , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/genética , Estrellas de Mar , Factores de Tiempo , Ubiquitina-Proteína Ligasas , XenopusRESUMEN
Ca(2+) channel inactivation is a key element in controlling the level of Ca(2+) entry through voltage-gated Ca(2+) channels. Interaction between the pore-forming alpha(1) subunit and the auxiliary beta subunit is known to be a strong modulator of voltage-dependent inactivation. Here, we demonstrate that an N-terminal membrane anchoring site (MAS) of the beta(2a) subunit strongly reduces alpha(1A) (Ca(V)2.1) Ca(2+) channel inactivation. This effect can be mimicked by the addition of a transmembrane segment to the N terminus of the beta(2a) subunit. Inhibition of inactivation by beta(2a) also requires a link between MAS and another important molecular determinant, the beta interaction domain (BID). Our data suggest that mobility of the Ca(2+) channel I-II loop is necessary for channel inactivation. Interaction of this loop with other identified intracellular channel domains may constitute the basis of voltage-dependent inactivation. We thus propose a conceptually novel mechanism for slowing of inactivation by the beta(2a) subunit, in which the immobilization of the channel inactivation gate occurs by means of MAS and BID.
Asunto(s)
Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Subunidades de Proteína , Animales , Bario/farmacología , Sitios de Unión/genética , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo Q/metabolismo , Línea Celular , Quelantes/farmacología , Electrofisiología , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/genética , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Transfección , XenopusRESUMEN
MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different classes. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. In this paper, we demonstrate that MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. We show that MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. We discuss these findings in the context of a universal mechanism for meiotic maturation in oocytes throughout the animal kingdom.
Asunto(s)
Factor Promotor de Maduración/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Xenopus/crecimiento & desarrollo , Xenopus/metabolismo , Animales , Benzoquinonas , Proteína Quinasa CDC2/metabolismo , Ciclina B/metabolismo , Fosfatasa 6 de Especificidad Dual , Activación Enzimática/efectos de los fármacos , Femenino , Insulina/farmacología , Lactamas Macrocíclicas , Meiosis/efectos de los fármacos , Meiosis/fisiología , Oocitos/efectos de los fármacos , Progesterona/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Quinonas/farmacologíaRESUMEN
During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Activación del Canal Iónico , Secuencia de Bases , Canales de Calcio/genética , Cartilla de ADN , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
X-PAKs are involved in negative control of the process of oocyte maturation in Xenopus (). In the present study, we define more precisely the events targetted by the kinase in the inhibition of the G2/M transition. We show that microinjection of recombinant X-PAK1-Cter active kinase into progesterone-treated oocytes prevents c-Mos accumulation and activation of both MAPK and maturation-promoting factor (MPF). In conditions permissive for MAPK activation, MPF activation still fails. We demonstrate that a constitutive truncated version of X-PAK1 (X-PAK1-Cter) does not prevent the association of cyclin B with p34(cdc2) but rather prevents the activation of the inactive complexes present in the oocyte. Proteins participating in the MPF amplification loop, including the Cdc25-activating Polo-like kinase are all blocked. Indeed, using active MPF, the amplification loop is not turned on in the presence of X-PAK1. Our results indicate that X-PAK and protein kinase A targets in the control of oocyte maturation are similar and furthermore that this negative regulation is not restricted to meiosis, because we demonstrate that G2/M progression is also prevented in Xenopus cycling extracts in the presence of active X-PAK1.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Fase G2 , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Catálisis , Proteínas de Ciclo Celular/antagonistas & inhibidores , Activación Enzimática , Femenino , Oocitos/efectos de los fármacos , Oocitos/enzimología , Progesterona/farmacología , Proteínas Recombinantes/metabolismo , Xenopus , Quinasas p21 ActivadasRESUMEN
We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.
Asunto(s)
Fase G2/fisiología , Factor Promotor de Maduración/metabolismo , Mitosis/fisiología , Óvulo/enzimología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Xenopus , Fosfatasas cdc25 , Animales , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/fisiología , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Activación Enzimática , Meiosis , Óvulo/citología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , XenopusRESUMEN
Micro-injection of catalytically inactive GST-cdc2-K33R or GST-cdk2-K33R fusion proteins, each of which efficiently titrates cyclin B in oocytes and prevents assembly of cyclin B-cdc2 complexes, readily induces premature DNA replication in starfish oocytes after emission of the first polar body. Moreover, partial ablation of cyclin B mRNA by micro-injection of antisense oligonucleotides facilitates premature DNA replication induced by the dominant-negative cdc2 and cdk2 mutant proteins. We thus propose that enhanced translation of cyclin B after GVBD, a universal feature of oocyte maturation in the animal kingdom, and subsequent assembly of cyclin B-cdc2 complexes, are part of the checkpoint that prevents DNA replication in the oocyte after emission of the first polar body. MAPK inactivation is neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation. However, micro-injection of a N-terminally truncated form of the budding yeast STE11 protein, that constitutively maintains MAPK active after the second meiotic cleavage, prevents fertilized eggs from proceeding into embryogenesis, and arrests them at G2, as is the case in unfertilized eggs that cannot inactivate MAPK after the second meiotic cleavage. We thus propose that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development.
Asunto(s)
Proteína Quinasa CDC2/fisiología , Ciclinas/fisiología , Replicación del ADN/fisiología , Meiosis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Oocitos/fisiología , Conejos , Transducción de Señal , Estrellas de MarRESUMEN
A few years after the identification of cyclin B-cdc2 kinase as the universal factor that controls onset of M-phase in eukaryotic cells, MPF (M-phase promoting factor), it became evident that all transitions of the cell cycle are controlled through phosphorylation of specific targets due to changes in the activity of a variety of cyclin-dependent kinases (cdks). These transitions include conversion of quiescent cells to a state of active proliferation, commitment to DNA replication, initiation of DNA replication, and entry into and exit from mitosis. Changes in the activity of cdks along the cell cycle depend not only on their association with a variety of cyclins (including G1/S and G2/M cyclins) and on posttranslational modifications by phosphorylation-dephosphorylation reactions, but also on specific protein inhibitors and on protein degradation.
Asunto(s)
División Celular/fisiología , Quinasas Ciclina-Dependientes/fisiología , Animales , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Replicación del ADN , Humanos , MitosisRESUMEN
Transitions of the cell cycle are controlled by cyclin-dependent protein kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A kinase responsible for this specific phosphorylation, called CAK for cdk-activating kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.
Asunto(s)
Quinasas Ciclina-Dependientes , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Núcleo Celular/metabolismo , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 5 , Clonación Molecular , ADN Complementario , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Xenopus , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
In most animals, the rate of cyclin B synthesis increases after nuclear envelope breakdown during the first meiotic cell cycle. We have found that cyclin B-cdc2 kinase activity drops earlier in emetine-treated than in control starfish oocytes, although the protein synthesis inhibitor does not activate the cyclin degradation pathway prematurely. Moreover, protein synthesis is required to prevent meiotic cleavage to occur prematurely, sometimes before chromosomes have segregated on the metaphase plate. In normal conditions, increased synthesis of cyclin B after germinal vesicle breakdown (GVBD) balances cyclin degradation and increases the time required for cyclin B-cdc2 kinase to drop below the level that inhibits cleavage. Taken together, these results point to cyclin B as a possible candidate that could explain the need for increased protein synthesis during meiosis I. Although direct experimental evidence was not provided in the present work, cyclin B synthesis after GVBD may be important for correct segregation of homologous chromosomes at the end of first meiotic metaphase, as shown by a variety of cytological disorders that accompany premature cleavage. Although the overall stimulation of protein synthesis because of cdc2 kinase activation is still observed in oocytes from which the germinal vesicle has been removed before hormonal stimulation, the main increase of cyclin B synthesis normally observed after germinal vesicle breakdown is suppressed. The nuclear factor required for specific translation of cyclin B after GVBD is not cyclin B mRNA, as shown by using a highly sensitive reverse transcription followed by polymerase chain reaction procedure that failed to detect any cyclin B mRNA in isolated germinal vesicles.
Asunto(s)
Factores Biológicos/fisiología , Ciclinas/biosíntesis , Proteínas del Huevo/biosíntesis , Regulación de la Expresión Génica , Meiosis , Oocitos/fisiología , Biosíntesis de Proteínas , Anafase , Animales , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Ciclinas/genética , Ciclinas/fisiología , Proteínas del Huevo/genética , Emetina/farmacología , Microtúbulos/efectos de los fármacos , Microtúbulos/ultraestructura , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , ADN Polimerasa Dirigida por ARN , Estrellas de MarRESUMEN
Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues).
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Quinasas Ciclina-Dependientes , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , Treonina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Activación Enzimática/genética , Guanosina Trifosfato/metabolismo , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Estrellas de Mar , Xenopus , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
A method to determine the humidity-conditioned gravimetric water content of hydrogel contact lens materials has been developed, in which errors due to blotting have been eliminated by conditioning the lens in a series of relative humidity (RH) environments before measuring the water content gravimetrically, and then extrapolating the water content to 100% RH. This method has been used to determine the water contents of representative materials from each of the four FDA lens groups, which were compared with their labeled values, as well as with values obtained from refractive index measurements. The deviation of the water content of soft contact lenses as measured by refractive index from that obtained gravimetrically increased as the water content decreased. The humidity-conditioned gravimetric method to determine water content of hydrophilic contact lenses is being proposed as an International Organization for Standardization (ISO) standard, as an improvement over the gravimetric and refractive index methods.
Asunto(s)
Lentes de Contacto Hidrofílicos , Humedad , Polietilenglicoles , Agua/análisis , Caprolactama/análogos & derivados , Gravitación , Hidrogel de Polietilenoglicol-Dimetacrilato , Polímeros , RefractometríaRESUMEN
PP63 is a liver specific phosphorylated glycoprotein encoded by a single copy gene, which has the property of inhibiting both autophosphorylation and tyrosine kinase activity of the insulin receptor. In this study, we have analyzed the structure activity relationship of the pp63 gene promoter. Five protein binding sites were found in the proximal 5' flanking region of the gene (-223 to +4). Using oligonucleotides as competitors and purified recombinant C/EBP in footprinting and gel retardation assays, we identified two typical C/EBP sites (X1 and X3) plus a heterogenous, C/EBP-NF1 like site (X5), separated by two classical NF1 binding sites (X2 and X4). C/EBP or the related proteins were predominantly involved in supporting cell-free transcription. Occupancy of the first high affinity C/EBP site conferred almost maximal promoter efficiency, in vitro. However, this pp63 promoter activity remained very low as compared to that in intact hepatocytes. In these cells, occupancy of the first C/EBP (X1) and NF1 (X2) sites was already required for achieving a weak transcriptional activity. The use of the second C/EBP site (X3) strongly enhanced transcription, up to 60-70% of the maximum, whereas occupancy of the two more distal sites (X4 and X5) was necessary to fully activate the promoter. Thus, the strength of the promoter as well as the liver specific expression of pp63 gene appear to result from the interplay of several DNA-protein complexes involving mainly C/EBP and/or related proteins as well as the ubiquitous NF1 factor(s), rather than from the interaction of a more liver specific trans-acting factor with the promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Glicoproteínas/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteínas Potenciadoras de Unión a CCAAT , Sistema Libre de Células , ADN/metabolismo , Masculino , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Endogámicas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Transcripción Genética/genética , alfa-2-Glicoproteína-HSRESUMEN
Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Ciclinas/metabolismo , Meiosis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Autorradiografía , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Inmunohistoquímica , Metafase , Oocitos/citología , Proteínas Proto-Oncogénicas c-mos , XenopusRESUMEN
Cilia may be carried into the eye following a penetrating injury. Such a case of a cilium in the anterior chamber is described. The cilium was discovered years later following an injury. A cilium was noted to extend through the cornea into the anterior chamber barely touching the anterior lens capsule. Although the lens surface directly directly adjacent to the foreign body was clear, an anterior capsule lens opacification and area of posterior syncechia were noted at the 3 o'clock meridian. The eye was quiet with no cell or flare noted. The vision was 20/20.