RESUMEN
BACKGROUND: Specific guidelines to manage caesarean delivery anaesthesia are lacking. A European multicentre study, ACCESS investigates caesarean delivery anaesthesia management in European centres. In order to identify ACCESS participating centres, a registration survey was created. OBJECTIVE: The aim of the current report is to describe the characteristics of ACCESS study participating centres, the rationale for the ACCESS study and the study methodology. DESIGN AND SETTING: The ACCESS study is a European multicentre cross-sectional study to describe anaesthesia management for caesarean delivery (CD) using a snapshot (2-week) design. The ACCESS registration survey gathered: contact details for National Coordinators (NC); Lead Investigators (LI) per centre; centre annual CD volume; expected no. of CD during 2-week snapshot window; centre practice information; data collection language. The ACCESS registration survey was launched July 2022 (Google Forms, Google Inc., Mountain View, CA, USA) and distributed through personal connections, national and international societies, social media networks, during Euroanaesthesia 2023, through the European Society of Anaesthesiology and Intensive Care newsletter. RESULTS: The ACCESS registration survey identified Lead Investigators for 418 centres, in 32 countries, representing an anticipated number of 15,073 CD cases over the planned 12-month study period. A median (range) of 20 (2 to 400) CD cases are anticipated per centre during the 2-week snapshot window. Most 366/418 (87.6%) centres are small, ≤2000 annual CD cases, 42 are medium 2000-5000 cases and 10 are large, ≥5000 annual CD cases. Registered centres reported in 134 (32.0%) centres that anaesthesia for caesarean delivery is performed mostly by a specialist obstetric anaesthesiologist. CONCLUSION: The ACCESS registration survey revealed variability in volume and CD practice as well as training-levels and staffing among European countries. The ACCESS study (https://www.access-study.org/) aims to generate practice data to guide CD anaesthetic management strategies.
Asunto(s)
Anestesia Obstétrica , Cesárea , Humanos , Cesárea/estadística & datos numéricos , Femenino , Estudios Transversales , Embarazo , Anestesia Obstétrica/estadística & datos numéricos , Anestesia Obstétrica/métodos , Europa (Continente) , Encuestas y CuestionariosRESUMEN
Models of contact tracing often over-simplify the effects of quarantine and isolation on disease transmission. We develop a model that allows us to investigate the importance of these factors in reducing the effective reproduction number. We show that the reduction in onward transmission during quarantine and isolation has a bigger effect than tracing coverage on the reproduction number. We also show that intuitively reasonable contact tracing performance indicators, such as the proportion of contacts quarantined before symptom onset, are often not well correlated with the reproduction number. We conclude that provision of support systems to enable people to quarantine and isolate effectively is crucial to the success of contact tracing.
Asunto(s)
COVID-19/transmisión , Trazado de Contacto/métodos , Número Básico de Reproducción , COVID-19/metabolismo , Trazado de Contacto/estadística & datos numéricos , Brotes de Enfermedades , Humanos , Modelos Teóricos , Aislamiento de Pacientes , Cuarentena/métodos , Cuarentena/psicología , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Aislamiento Social/psicologíaRESUMEN
We use a stochastic branching process model, structured by age and level of healthcare access, to look at the heterogeneous spread of COVID-19 within a population. We examine the effect of control scenarios targeted at particular groups, such as school closures or social distancing by older people. Although we currently lack detailed empirical data about contact and infection rates between age groups and groups with different levels of healthcare access within New Zealand, these scenarios illustrate how such evidence could be used to inform specific interventions. We find that an increase in the transmission rates among children from reopening schools is unlikely to significantly increase the number of cases, unless this is accompanied by a change in adult behaviour. We also find that there is a risk of undetected outbreaks occurring in communities that have low access to healthcare and that are socially isolated from more privileged communities. The greater the degree of inequity and extent of social segregation, the longer it will take before any outbreaks are detected. A well-established evidence for health inequities, particularly in accessing primary healthcare and testing, indicates that Maori and Pacific peoples are at a higher risk of undetected outbreaks in Aotearoa New Zealand. This highlights the importance of ensuring that community needs for access to healthcare, including early proactive testing, rapid contact tracing and the ability to isolate, are being met equitably. Finally, these scenarios illustrate how information concerning contact and infection rates across different demographic groups may be useful in informing specific policy interventions.
Asunto(s)
COVID-19/transmisión , Disparidades en Atención de Salud , Modelos Biológicos , Pandemias , SARS-CoV-2 , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Número Básico de Reproducción/estadística & datos numéricos , COVID-19/epidemiología , COVID-19/prevención & control , Niño , Simulación por Computador , Trazado de Contacto , Femenino , Accesibilidad a los Servicios de Salud/estadística & datos numéricos , Disparidades en Atención de Salud/estadística & datos numéricos , Humanos , Masculino , Conceptos Matemáticos , Persona de Mediana Edad , Nueva Zelanda/epidemiología , Pandemias/prevención & control , Pandemias/estadística & datos numéricos , Procesos Estocásticos , Adulto JovenRESUMEN
Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.
Asunto(s)
Lamina Tipo B , Proteínas Nucleares/química , Animales , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Calcio/farmacología , Pollos , Cristalización , Escherichia coli/genética , Escherichia coli/metabolismo , Laminas , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Fosforilación , Polímeros , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain-specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed alpha-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.
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Retículo Endoplásmico/metabolismo , Proteínas de la Membrana , Fosfoproteínas/metabolismo , Transactivadores , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Membrana Nuclear/metabolismo , Fosfoproteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.
Asunto(s)
Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero/genética , Compartimento Celular , Núcleo Celular/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Sustancias Macromoleculares , Proteínas Nucleares/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citología , Solubilidad , Relación Estructura-Actividad , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
The yeast protein RAP1, initially described as a transcriptional regulator, binds in vitro to sequences found in a number of seemingly unrelated genomic loci. These include the silencers at the transcriptionally repressed mating-type genes, the promoters of many genes important for cell growth, and the poly[(cytosine)1-3 adenine] [poly(C1-3A)] repeats of telomeres. Because RAP1 binds in vitro to the poly(C1-3A) repeats of telomeres, it has been suggested that RAP1 may be involved in telomere function in vivo. In order to test this hypothesis, the telomere tract lengths of yeast strains that contained conditionally lethal (ts) rap1 mutations were analyzed. Several rap1ts alleles reduced telomere length in a temperature-dependent manner. In addition, plasmids that contain small, synthetic telomeres with intact or mutant RAP1 binding sites were tested for their ability to function as substrates for poly(C1-3A) addition in vivo. Mutations in the RAP1 binding sites reduced the efficiency of the addition reaction.
Asunto(s)
Cromosomas Fúngicos/ultraestructura , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Genes del Tipo Sexual de los Hongos , Saccharomyces cerevisiae/genética , Factores de Transcripción , Secuencia de Bases , Sitios de Unión , Cromosomas Fúngicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Mutación , Plásmidos , Poli A/metabolismo , Poli C/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Temperatura , Transformación GenéticaRESUMEN
In the yeast Saccharomyces cerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres. The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Animales , Nucléolo Celular , Cromatina , Reparación del ADN , Replicación del ADN , Proteínas Fúngicas/metabolismo , Humanos , Ligandos , Telómero , Ubiquitinas/metabolismoRESUMEN
A ribosomal frameshift is required for the synthesis of an essential component of the yeast telomerase pathway; this and other findings on telomerases from many species raise interesting questions regarding the evolutionary relationship between telomerases and retrotransposons lacking long terminal repeats.
Asunto(s)
ADN/fisiología , Retroelementos/fisiología , Telomerasa/fisiología , Animales , Análisis Mutacional de ADN , Evolución Molecular , Mutación del Sistema de Lectura , Humanos , Sistemas de Lectura AbiertaRESUMEN
The Ku heterodimer, conserved in a wide range of eukaryotes, plays a multiplicity of roles in yeast. First, binding of Ku, which is composed of a 70 kDa (Hdf1p) and an 80 kDa (Hdf2p) subunit [1-3], to double-strand breaks promotes non-homologous end-to-end joining of DNA [3]. Second, Ku appears to participate in DNA replication, regulating both the number of rounds of replication permissible within the cell cycle and the structure of the initiation complex [3,4]. Furthermore, mutations in HDF1 or HDF2 rapidly reduce telomeric poly (TG1-3) tract size [1-3], hinting also at a possible telomeric function of Ku. We show here that the two subunits of the Ku heterodimer play a key role in maintaining the integrity of telomere structure. Mutations in either Ku subunit increased the single-strandedness of the telomere in a cell-cycle-independent fashion, unlike wild-type cells which form 3' poly(TG1-3) overhangs exclusively in late S phase [5]. In addition, mutations enhanced the instability of elongated telomeres to degradation and recombination. Both Ku subunits genetically interacted with the putative single-stranded telomere-binding protein Cdc13p. We propose that Ku protects the telomere against nucleases and recombinases.
Asunto(s)
Antígenos Nucleares , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Recombinación Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Telómero/metabolismo , Secuencia de Bases , Cromosomas Fúngicos , Cruzamientos Genéticos , Ciclina B/química , Ciclina B/genética , Ciclina B/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Heterocigoto , Autoantígeno Ku , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Oligodesoxirribonucleótidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/fisiología , Telómero/genética , Telómero/ultraestructura , Factores de Transcripción/metabolismoRESUMEN
We have previously identified a process in the yeast Saccharomyces cerevisiae that results in the contraction of elongated telomeres to wild-type length within a few generations. We have termed this process telomeric rapid deletion (TRD). In this study, we use a combination of physical and genetic assays to investigate the mechanism of TRD. First, to distinguish among several recombinational and nucleolytic pathways, we developed a novel physical assay in which HaeIII restriction sites are positioned within the telomeric tract. Specific telomeres were subsequently tested for HaeIII site movement between telomeres and for HaeIII site retention during TRD. Second, genetic analyses have demonstrated that mutations in RAD50 and MRE11 inhibit TRD. TRD, however, is independent of the Rap1p C-terminal domain, a central regulator of telomere size control. Our results provide evidence that TRD is an intrachromatid deletion process in which sequences near the extreme terminus invade end-distal sequences and excise the intervening sequences. We propose that the Mre11p-Rad50p-Xrs2p complex prepares the invading telomeric overhang for strand invasion, possibly through end processing or through alterations in chromatin structure.
Asunto(s)
Cromátides/genética , Endodesoxirribonucleasas , Exodesoxirribonucleasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros , Telómero/genética , Factores de Transcripción , ADN/genética , Proteínas de Unión al ADN/fisiología , Desoxirribonucleasas de Localización Especificada Tipo II/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Modelos Genéticos , Mutación , Recombinación Genética , Eliminación de Secuencia , Complejo Shelterina , Intercambio de Cromátides HermanasRESUMEN
The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.
Asunto(s)
Proteínas de Unión al GTP/genética , Saccharomyces cerevisiae/genética , Telómero , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Deleción Cromosómica , ADN de Hongos , Proteínas de Unión al GTP/metabolismo , Datos de Secuencia Molecular , Mutación , No Disyunción Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rapRESUMEN
Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.
Asunto(s)
Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Serina Endopeptidasas , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Telómero , Transactivadores/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cromosomas Fúngicos , Proteínas Fúngicas/biosíntesis , Proteínas de Unión al GTP/metabolismo , Genotipo , Modelos Estructurales , Mutagénesis , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Supresión Genética , Transactivadores/biosíntesis , Transcripción Genética , Proteínas de Unión al GTP rapRESUMEN
The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.
Asunto(s)
Núcleo Celular/análisis , Proteínas Fúngicas/genética , Empalme del ARN , Proteínas de Saccharomyces cerevisiae , Levaduras/genética , Anticuerpos Antifúngicos/inmunología , Secuencia de Bases , Núcleo Celular/inmunología , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/análisis , Proteínas Fúngicas/inmunología , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Proteínas de Unión al ARN , Temperatura , Levaduras/inmunologíaRESUMEN
Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.
Asunto(s)
Antígenos Virales de Tumores/fisiología , Transformación Celular Viral , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Tirosina/análogos & derivados , Técnicas de Inmunoadsorción , Peso Molecular , Fosfotirosina , Poliomavirus , Proteínas Tirosina Fosfatasas , Tirosina/metabolismo , Vanadio/farmacologíaRESUMEN
The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.
Asunto(s)
Virus del Sarcoma Aviar/genética , Genes Virales , Genes , Proteínas Quinasas/genética , Proteínas de los Retroviridae/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Virus del Sarcoma Aviar/enzimología , Escherichia coli/genética , Cinética , Proteína Oncogénica pp60(v-src) , Fosforilación , Plásmidos , Biosíntesis de Proteínas , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The yeast nucleoporins Nsp1p, Nup49p, and Nup57p form a complex at the nuclear pores which is involved in nucleocytoplasmic transport. To investigate the molecular basis underlying complex formation, recombinant full-length Nup49p and Nup57p and the carboxyl-terminal domain of Nsp1p, which lacks the FXFG repeat domain, were expressed in Escherichia coli. When the three purified proteins were mixed together, they spontaneously associated to form a 150-kDa complex of 1:1:1 stoichiometry. In this trimeric complex, Nup57p fulfills the role of an organizing center, to which Nup49p and Nsp1p individually bind. For this interaction to occur, only two heptad repeat regions of the Nsp1p carboxyl-terminal domain are required, each region being about 50 amino acids in length. Finally, the reconstituted complex has the capability to bind to full-length Nic96p but not to mutant forms which also do not interact in vivo. When added to permeabilized yeast cells, the complex associates with the nuclear envelope and the nuclear pores. We conclude that Nsp1p, Nup49p, and Nup57p can reconstitute a complex in vitro which is competent for further assembly with other components of nuclear pores.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Sitios de Unión , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Electrónica , Microscopía Fluorescente , Proteínas Nucleares/química , Proteínas Nucleares/genética , Permeabilidad , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Levaduras/metabolismoRESUMEN
A rapidly developing dynamical gradient can be formed in the analytical centrifuge when a buffer solution prepared in D2O is underlayered under the same buffer solution prepared in H2O in a specially designed double sector cell. In a short time the boundary layer spreads to form the gradient. Heavy particles (S greater than or equal to 10S) will band in the gradient corresponding to their density, which can be determined accurately. To this end, the buffer may contain density adjusting additives such as sucrose. We present results with this technique for lipid vesicles, for a Ca-antagonist bound to vesicles, as well as for a lipoprotein and reconstituted regular membrane protein-lipid arrays.