RESUMEN
Herein, we present a series of five tetrabutylammonium (TBA) sulfonate-urea amphiphilic salts. In solution these amphiphilic salts have been shown to form a variety of self-associated species. The proportion and type of which are both solvent and concentration dependent. In DMSO-d6 a variety of NMR experiments provide evidence towards the formation of mainly dimeric over larger aggregate species. Increasing the percentage of water was shown to increase the concentration of the larger aggregates over dimers in solution. A correlation was established between critical micelle concentration (CMC) values obtained in a 1 : 19 EtOH : H2O mixture, dimeric self-association constants obtained in a DMSO-d6 - 0.5% H2O and the results of simple semi-empirical PM6 computational modelling methods. This approach begins to quantify the role of hydrogen bonding in amphiphile self-association and the effects it imparts on surfactant properties. This consequently provides preliminary evidence that these properties maybe predicted by simple low level computational modelling techniques.
RESUMEN
The Dbf2 protein kinase functions as part of the mitotic-exit network (MEN), which controls the inactivation of the Cdc28-Clb2 kinase in late mitosis [1]. The MEN includes the Tem1 GTP binding protein; the kinases Cdc15 and Cdc5; Mob1, a protein of unknown function; and the phosphatase Cdc14 [2]. Here we have used Dbf2 kinase activity to investigate the regulation and order of function of the MEN. We find that Tem1 acts at the top of the pathway, upstream of Cdc15, which in turn functions upstream of Mob1 and Dbf2. The Cdc5 Polo-like kinase impinges at least twice on the MEN since it negatively regulates the network, probably upstream of Tem1, and is also required again for Dbf2 kinase activation. Furthermore, we find that regulation of Dbf2 kinase activity and actin ring formation at the bud neck are causally linked. In metaphase-arrested cells, the MEN inhibitor Bub2 restrains both Dbf2 kinase activity [3] and actin ring formation [4]. We find that the MEN proteins that are required for Dbf2 kinase activity are also required for actin ring formation. Thus, the MEN is crucial for the regulation of cytokinesis, as well as mitotic exit.
Asunto(s)
Proteínas Fúngicas/fisiología , Mitosis/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiologíaRESUMEN
Activation of Cdc2, is the universal event controlling the onset of mitosis. In higher eukaryotes, Cdc2 activity is in part regulated by inhibitory phosphorylation of Thr14 and Tyr15, catalyzed by Wee1 and Myt1, which prevents catastrophic premature entry into mitosis. In this study we defined the function of Myt1 by overexpression studies in both S. pombe and a human osteosarcoma cell line. Similar to Wee1, overexpression of human Myt1 prevented entry into mitosis in both cell types; however, Myt1 catalytic activity was not essential for the cell cycle delay observed with human cells. Myt1 expression was restricted to proliferating cells. Furthermore, we detected no major decline in Myt1 protein abundance prior to the entry into mitosis, which coincides with the loss of Myt1 activity. We localized mitotic phosphoepitopes, recognized by the monoclonal antibody MPM-2, to the C-terminal domain of Myt1. The mitotic peptidyl-prolyl isomerase, Pin1, was able to associate with this domain in a phosphorylation-dependent manner. Truncation of the C-terminal domain of Myt1 prevented its ability to induce G(2)/M phase arrest in overexpression studies in human cells and dramatically reduced its ability to phosphorylate Cdc2 in vitro. We demonstrate that the C-terminal domain of Myt1 was required for recruitment of Cdc2, and we infer that this domain lies in the cytoplasm because it can interact with and is phosphorylated by Cdc2. In conclusion, we propose that Myt1 can negatively regulate Cdc2/cyclin B1 and inhibit G(2)/M progression by two means, both of which require the C-terminal domain; first, Myt1 can bind and sequester Cdc2/cyclin B1 in the cytoplasm preventing entry into the nucleus, and, second, it can phosphorylate associated Cdc2/cyclin B1 at Thr14 and Tyr15 thus inhibiting its catalytic activity.
Asunto(s)
Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/metabolismo , Fase G2/fisiología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Anticuerpos Monoclonales , Biomarcadores , Dominio Catalítico/genética , Activación Enzimática/fisiología , Epítopos/inmunología , Regulación Fúngica de la Expresión Génica , Humanos , Proteínas de la Membrana , Mitosis/fisiología , Mutagénesis Sitio-Dirigida/fisiología , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/metabolismo , Fosfoproteínas/inmunología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimologíaRESUMEN
We have characterized HsCdc6, a human protein homologous to the budding yeast Cdc6p that is essential for DNA replication. We show that, unlike Cdc6p, the levels of HsCdc6 protein remain constant throughout the cell cycle in human cells. However, phosphorylation of HsCdc6 is regulated during the cell cycle. HsCdc6 is an excellent substrate for Cdk2 in vitro and is phosphorylated in vivo at three sites (Ser-54, Ser-74, and Ser-106) that are phosphorylated by Cdk2 in vitro, strongly suggesting that HsCdc6 is an in vivo Cdk substrate. HsCdc6 is nuclear in G1, but translocates to the cytoplasm at the start of S phase via Crm1-dependent export. An HsCdc6A1A2A3 mutant, which mimics unphosphorylated HsCdc6, is exclusively nuclear, and its expression inhibits initiation of DNA replication. An HsCdc6E1E2E3 mutant, which mimics phosphorylated HsCdc6, is exclusively cytoplasmic and is not associated with the chromatin/nuclear matrix fraction. Based on these results, we propose that phosphorylation of HsCdc6 by Cdks regulates DNA replication of at least two steps: first, by promoting initiation of DNA replication and, second, through nuclear exclusion preventing DNA rereplication.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Anticuerpos , Proteínas de Ciclo Celular/química , Línea Celular , Células Cultivadas , Cromatina/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Células HeLa , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Matriz Nuclear/fisiología , Proteínas Nucleares/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fosforilación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Piel/citologíaAsunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Electroforesis en Gel Bidimensional/métodos , Mapeo Peptídico/métodos , Fosfopéptidos/análisis , Isótopos de Fósforo/metabolismo , Pruebas de Precipitina/métodos , Tripsina/metabolismo , Antígenos de Neoplasias , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Células HeLa , HumanosRESUMEN
Topoisomerase II is an essential enzyme in all organisms with several independent roles in DNA metabolism. In this article we review our knowledge on the regulation of the expression and catalytic activity of topoisomerase II in both lower and higher eukaryotes. Current data indicate that the regulation of topoisomerase II gene expression is complex, with positive and negative controls in evidence at the level of both promoter activity and mRNA stability. Similarly, the activity of the mature enzyme can be regulated by the action of several different protein kinases. Of particular interest is the cell cycle-dependent phosphorylation of topoisomerase II, including multiple, mitosis-specific modifications, which are proposed to regulate the essential chromosome decatenation activity of the enzyme.
Asunto(s)
ADN-Topoisomerasas de Tipo II/fisiología , Regulación Enzimológica de la Expresión Génica/genética , Animales , Secuencia de Bases , Ciclo Celular/fisiología , ADN/metabolismo , Células Eucariotas , Mamíferos , Datos de Secuencia Molecular , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Análisis de Secuencia de ADN , Transcripción Genética/genéticaRESUMEN
TGF-beta and activin induce the phosphorylation and activation of Smad2 and Smad3, but how these proteins stimulate gene transcription is poorly understood. We report that TGF-beta receptor phosphorylation of Smad3 promotes its interaction with the paralogous coactivators CBP and p300, whereas CBP/p300 binding to nonphosphorylated Smad3 or its oligomerization partner Smad4 is negatively regulated by Smad-intramolecular interactions. Furthermore, p300 and TGF-beta receptor-phosphorylated Smad3 synergistically augment transcriptional activation. Thus, CBP/p300 are important components of activin/TGF-beta signaling and may mediate the antioncogenic functions of Smad2 and Smad4.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteína de Unión a CREB , Línea Celular Transformada , Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Fosforilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Smad2 , Proteína smad3 , Proteína Smad4 , Transactivadores/genética , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasRESUMEN
Previous reports have indicated that topoisomerase II (topo II) co-purifies with and is a substrate for casein kinase II. We have carried out a detailed study of the effect that purified casein kinase II has on the activity of purified recombinant human topo IIalpha. Co-incubation of topo IIalpha and casein kinase II led to an apparent activation of the topo IIalpha; however, in experiments in which topo IIalpha was preincubated at 37 degrees C with or without native casein kinase II prior to assaying for decatenation activity, it emerged that the kinase was exerting its "activating" function via a decrease in the rate of topo IIalpha enzyme inactivation during the incubation period. This stabilization of topo IIalpha by casein kinase II was ATP-independent and was observed in both mutated and truncated derivatives of topo IIalpha lacking the major casein kinase II phospho-acceptor sites, indicating the lack of a requirement for phosphorylation. Consistent with a nonenzymatic role for casein kinase II, stoichiometric quantities of kinase were required for topo IIalpha stabilization. These data indicate that casein kinase II plays a significant role in regulating human topo IIalpha protein action via stabilization against thermal inactivation.
Asunto(s)
ADN-Topoisomerasas de Tipo II/metabolismo , Isoenzimas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Antígenos de Neoplasias , Quinasa de la Caseína II , Núcleo Celular/enzimología , Proteínas de Unión al ADN , Estabilidad de Enzimas , Células HeLa , Humanos , Isoenzimas/antagonistas & inhibidores , Fosforilación , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Inhibidores de Topoisomerasa IIRESUMEN
We have identified a novel human protein, PRC1, that is involved in cytokinesis. PRC1 is a good substrate for several CDKs in vitro and is phosphorylated in vivo at sites that are phosphorylated by CDK in vitro, strongly suggesting that PRC1 is an in vivo CDK substrate. PRC1 has sequence homology to the budding yeast anaphase spindle elongation factor Ase1p. Like Ase1p, PRC1 protein levels are high during S and G2/M and drop dramatically after cells exit mitosis and enter G1. PRC1 is a nuclear protein in interphase, becomes associated with mitotic spindles in a highly dynamic manner during mitosis, and localizes to the cell mid-body during cytokinesis. Microinjection of anti-PRC1 antibodies into HeLa cells blocked cellular cleavage, but not nuclear division, indicating a functional role for PRC1 in the process of cytokinesis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Clonación Molecular , Quinasas Ciclina-Dependientes/metabolismo , ADN Complementario/análisis , ADN Complementario/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Huso Acromático/metabolismo , Especificidad por Sustrato , Treonina/genéticaRESUMEN
OBJECTIVE: To compare an alternative treatment for lower extremity burns with the standard in-hospital treatment, in an attempt to shorten hospital stay. DESIGN: A case-control series. SETTING: A university-affiliated hospital. PATIENTS: All patients with a burn isolated to a lower extremity were treated over an 8-month period with split-thickness skin grafting (STSG), Unna paste dressing, immediate mobilization and early discharge. This group was compared with matched controls from the preceding 8 years treated with STSG, occlusive burn gauze dressing, bed rest and hospitalization. MAIN OUTCOME MEASURES: Duration of hospital stay and graft viability. RESULTS: Thirteen patients with an average wound size of 131 cm2 were treated with Unna paste and had a graft viability of greater than 95% and a burn-scar rating equivalent to that of patients treated with the earlier regimen. The duration of hospital stay decreased from a mean of 12.9 days to 1.4 days, with no complications. This translated into a saving of $10,350 per patient. CONCLUSIONS: This alternative treatment is safe, inexpensive and effective and is recommended as the treatment of choice for uncomplicated, noncircumferential lower extremity burns.
Asunto(s)
Quemaduras/terapia , Gelatina/uso terapéutico , Glicerol/uso terapéutico , Traumatismos de la Pierna/terapia , Compuestos de Zinc/uso terapéutico , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Control de Costos , Combinación de Medicamentos , Ambulación Precoz , Humanos , Tiempo de Internación , Persona de Mediana Edad , Apósitos Oclusivos , Trasplante de Piel , Supervivencia TisularRESUMEN
Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the alpha isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme. Here, we have shown that the enzymatic activity of topoisomerase II alpha protein purified from HeLa cell nuclei was strongly enhanced following phosphorylation by protein kinase C. We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alpha in HeLa cells. Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase II alpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G2/M phases of the cell cycle. To identify sites of phosphorylation, and the kinase(s) responsible for this modification, oligohistidine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coli and purified by affinity chromatography. Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase II alpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G2/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase II alpha protein. Site-directed mutagenesis studies indicated that phosphorylation of serine 29 generated both of these phosphopeptides. Our results implicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.
Asunto(s)
Ciclo Celular , ADN-Topoisomerasas de Tipo II/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , División Celular , Cartilla de ADN , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Fase G2 , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Mitosis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Mapeo Peptídico , Fosfopéptidos/química , Fosfopéptidos/aislamiento & purificación , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood. We have investigated whether or not the alpha isozyme of human topoisomerase II is phosphorylated in a cell-cycle phase-dependent manner. Two-dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and M phases. To identify the protein kinases involved in this cell-cycle phase-specific phosphorylation, oligohistidine-tagged recombinant domains of the topoisomerase II alpha protein were expressed in Escherichia coli, purified by affinity chromatography and phosphorylated in vitro by different protein kinases. Phosphorylation of the C-terminal domain of the topoisomerase II alpha protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites observed in vivo. The same phosphopeptides were obtained following phosphorylation of the C-terminal domain in vitro by the mitogen-activated protein kinase. Site-directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine-proline motif. Our data implicate one or more proline-directed kinases in the cell-cycle-dependent regulation of topoisomerase II alpha enzyme activity in human cells.
Asunto(s)
ADN-Topoisomerasas de Tipo II , ADN-Topoisomerasas de Tipo II/metabolismo , Fase G2 , Isoenzimas/metabolismo , Mitosis , Prolina/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN , Citometría de Flujo , Células HeLa , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Proteínas Recombinantes/metabolismoRESUMEN
Topoisomerase II enzymes play an essential role in human DNA metabolism. They are also recognized as primary targets of a number of anti-cancer drugs used in the treatment of breast cancer, which remains a leading cause of cancer-related death in women. While topoisomerase inhibitors have produced significant response rates in this disease, their use has been limited both by toxicity and by the development of resistance. In this article we review the extensive work which has not only increased our understanding of the biochemistry and molecular biology of type II topoisomerases but also enabled more rational drug design. Such knowledge should translate into increased clinical efficacy in the treatment of breast cancer and other malignancies.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Topoisomerasa II , Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/fisiología , Resistencia a Medicamentos , Femenino , HumanosRESUMEN
OBJECTIVE: To review recent experience with pilomatrixoma, particularly with respect to diagnosis. DESIGN: Chart review for an 8-year period. SETTING: A university-affiliated children's hospital. PATIENTS: Fifty children with 51 histologically proven pilomatrixomas. INTERVENTION: Excision of the lesion. MAIN OUTCOME MEASURES: Clinical presentation, diagnosis and pathological findings. RESULTS: The clinical presentation was typically that of an asymptomatic superficial mass that increased slowly in size and was located in the head and neck (78%) or chest and upper limbs. In one case the mass increased rapidly over 2 weeks and changed in colour from red to purple with areas of pink and tan. The referring diagnosis was incorrect in 94% of cases, and the preoperative diagnosis was incorrect in 57%. Pathologically the tumours were situated in the dermis or subcutaneous tissue. None were cystic, but several had areas of calcification. None of the tumours recurred after excision, but two patients had multiple lesions at different locations and times. CONCLUSIONS: Pilomatrixomas in children are simple to treat successfully but have variable clinical presentation. Early diagnosis is important so that unnecessary, aggressive surgery can be avoided.
Asunto(s)
Pilomatrixoma/cirugía , Neoplasias Cutáneas/cirugía , Niño , Humanos , Pilomatrixoma/diagnóstico , Pilomatrixoma/patología , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patologíaRESUMEN
Topoisomerase II protein is essential for cell proliferation and is known to exist as a phosphoprotein in cells from both lower and higher eukaryotic species. In this paper, we have investigated the phosphorylation of the alpha isozyme of human topoisomerase II. The topoisomerase II alpha protein was phosphorylated predominantly on serine residues in the human tumor cell lines HeLa and NSCLC-3. Two-dimensional tryptic phosphopeptide mapping studies revealed several sites of phosphorylation in vivo, including a major site that was common to topoisomerase II alpha protein from both HeLa and NSCLC-3 cells. To identify sites of phosphorylation, the regulatory C-terminal domain of human topoisomerase II alpha protein was overexpressed in Escherichia coli as a hexahistidine-tagged fusion protein and purified by nickel chelate chromatography. Tryptic phosphopeptide mapping revealed that casein kinase II phosphorylated the C-terminal domain primarily on 2 serine residues in vitro, which were shown to be sites of modification in vivo. Site-directed mutagenesis studies identified these casein kinase II-specific phosphorylation sites as serine 1524 and serine 1376.