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We analyzed a 6.7-yr span of data from a rotating torsion-pendulum containing ≈10^{23} polarized electrons to search for the "wind" arising from ultralight, axionlike dark matter with masses between 10^{-23} and 10^{-18} eV/c^{2}. Over much of this range we set a 95% confidence limit F_{a}/C_{e}>2×10^{15} eV on the axionlike decay constant.
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Background: Gene expression-based profiling of colorectal cancer (CRC) can be used to identify four molecularly homogeneous consensus molecular subtype (CMS) groups with unique biologic features. However, its applicability to colorectal premalignant lesions remains unknown. Patients and methods: We assembled the largest transcriptomic premalignancy dataset by integrating different public and proprietary cohorts of adenomatous and serrated polyps from sporadic (N = 311) and hereditary (N = 78) patient populations and carried out a comprehensive analysis of carcinogenesis pathways using the CMS random forest (RF) classifier. Results: Overall, transcriptomic subtyping of sporadic and hereditary polyps revealed CMS2 and CMS1 subgroups as the predominant molecular subtypes in premalignancy. Pathway enrichment analysis showed that adenomatous polyps from sporadic or hereditary cases (including Lynch syndrome) displayed a CMS2-like phenotype with WNT and MYC activation, whereas hyperplastic and serrated polyps with CMS1-like phenotype harbored prominent immune activation. Rare adenomas with CMS4-like phenotype showed significant enrichment for stromal signatures along with transforming growth factor-ß activation. There was a strong association of CMS1-like polyps with serrated pathology, right-sided anatomic location and BRAF mutations. Conclusions: Based on our observations made in premalignancy, we propose a model of pathway activation associated with CMS classification in colorectal carcinogenesis. Specifically, while adenomatous polyps are largely CMS2, most hyperplastic and serrated polyps are CMS1 and may transition into other CMS groups during evolution into carcinomas. Our findings shed light on the transcriptional landscape of premalignant colonic polyps and may help guide the development of future biomarkers or preventive treatments for CRC.
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Adenoma/diagnóstico , Biomarcadores de Tumor/genética , Pólipos del Colon/diagnóstico , Neoplasias Colorrectales/clasificación , Neoplasias Colorrectales/diagnóstico , Mutación , Lesiones Precancerosas/diagnóstico , Adenoma/genética , Pólipos del Colon/genética , Neoplasias Colorrectales/genética , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Fenotipo , Lesiones Precancerosas/genética , Valor Predictivo de las Pruebas , Pronóstico , TranscriptomaRESUMEN
BACKGROUND: Guidelines recommend the addition of a long-acting ß2-agonist (LABA) for patients whose asthma is uncontrolled on inhaled corticosteroid (ICS) monotherapy. For COPD patients the addition of an ICS to a long-acting bronchodilator is recommended for symptomatic patients at high risk of exacerbations. We examined whether in real-life practice guideline recommendations may delay optimal timing for initiation of combination treatment. METHODS: A modified Delphi process was undertaken with a panel of physicians, including six GPs and four pulmonologists, in practice in Germany. The first round comprised a semi-structured questionnaire, the second stage was an online discussion to reach consensus on 25 statements concerning the use of ICS/LABA in patients with asthma or COPD. RESULTS: Consensus was achieved on 24 of 25 prepared statements for early initiation of fixed ICS/LABA combination treatment. The panel agreed that a meaningful share of their asthma patients on ICS monotherapy experienced symptoms and exacerbations that should lead to addition of LABAs and that timely initiation of ICS/LABA therapy in asthma patients could improve asthma control, and prevent a significant number of emergency room visits, hospitalisations or additional specialist visits. The panel agreed that symptomatic patients with moderate to severe COPD, and frequent exacerbations should receive ICS without any delay in addition to their bronchodilator maintenance therapy. These patients could benefit from fewer exacerbations and a reduction in symptoms. The panel reached a consensus that fixed-dose ICS/LABA could have a positive effect on adherence, compared with separate inhalers for ICS and LABA, which may impact treatment outcomes. CONCLUSION: A panel of ten physicians working in primary and secondary care agreed on 24 out of 25 statements that supported the early initiation of fixed combination treatment, if indicated in a meaningful number of their asthma and COPD patients.
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Corticoesteroides/administración & dosificación , Agonistas Adrenérgicos beta/administración & dosificación , Asma/tratamiento farmacológico , Atención Primaria de Salud/normas , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Neumología/normas , Atención Secundaria de Salud/normas , Administración por Inhalación , Asma/complicaciones , Técnica Delphi , Combinación de Medicamentos , Alemania , Humanos , Guías de Práctica Clínica como Asunto , Enfermedad Pulmonar Obstructiva Crónica/complicacionesRESUMEN
We describe a liquid-cryogen free cryostat with ultra-low vibration levels, which allows for continuous operation of a torsion balance at cryogenic temperatures. The apparatus uses a commercially available two-stage pulse-tube cooler and passive vibration isolation. The torsion balance exhibits torque noise levels lower than room temperature thermal noise by a factor of about four in the frequency range of 3-10 mHz, limited by residual seismic motion and by radiative heating of the pendulum body. In addition to lowering thermal noise below room-temperature limits, the low-temperature environment enables novel torsion balance experiments. Currently, the maximum duration of a continuous measurement run is limited by accumulation of cryogenic surface contamination on the optical elements inside the cryostat.
RESUMEN
We describe a torsion pendulum with a large mass-quadrupole moment and a resonant frequency of 2.8 mHz, whose angle is measured using a Michelson interferometer. The system achieved noise levels of â¼200prad/Hz between 0.2 and 30 Hz and â¼10prad/Hz above 100 Hz. Such a system can be applied to a broad range of fields from the study of rotational seismic motion and elastogravity signals to gravitational wave observation and tests of gravity.
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Properly functioning on-site wastewater systems (OWS) are an integral component of the wastewater system infrastructure necessary to renovate wastewater before it reaches surface or ground waters. There are a large number of factors, including soil hydraulic properties, effluent quality and dispersal, and system design, that affect OWS function. The ability to evaluate these factors using a simulation model would improve the capability to determine the impact of wastewater application on the subsurface soil environment. An existing subsurface drip irrigation system (SDIS) dosed with sequential batch reactor effluent (SBRE) was used in this study. This system has the potential to solve soil and site problems that limit OWS and to reduce the potential for environmental degradation. Soil water potentials (Psi(s)) and nitrate (NO(3)) migration were simulated at 55- and 120-cm depths within and downslope of the SDIS using a two-dimensional code in HYDRUS-3D. Results show that the average measured Psi(s) were -121 and -319 cm, whereas simulated values were -121 and -322 cm at 55- and 120-cm depths, respectively, indicating unsaturated conditions. Average measured NO(3) concentrations were 0.248 and 0.176 mmol N L(-1), whereas simulated values were 0.237 and 0.152 mmol N L(-1) at 55- and 120-cm depths, respectively. Observed unsaturated conditions decreased the potential for NO(3) to migrate in more concentrated plumes away from the SDIS. The agreement (high R(2) values approximately 0.97) between the measured and simulated Psi(s) and NO(3) concentrations indicate that HYDRUS-3D adequately simulated SBRE flow and NO(3) transport through the soil domain under a range of environmental and effluent application conditions.
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Nitratos/química , Eliminación de Residuos Líquidos/instrumentación , Eliminación de Residuos Líquidos/métodos , Reactores Biológicos , Arquitectura y Construcción de Instituciones de Salud , Filtración , Modelos Teóricos , Nitrógeno/metabolismo , RestaurantesRESUMEN
This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial-S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.
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Flagelina/metabolismo , Inflamación/etiología , Mucosa Intestinal/microbiología , Salmonella typhimurium/patogenicidad , Línea Celular , Epitelio/inmunología , Epitelio/microbiología , Flagelina/genética , Humanos , Inflamación/inmunología , Inflamación/microbiología , Mediadores de Inflamación/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/inmunología , Modelos Biológicos , Mutación , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologíaRESUMEN
Two public beaches (Anderson and Hilton) in Newport News, Virginia, were frequently closed to swimming in 2004 due to high Enterococcus spp. counts that exceeded the regulatory standard. The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners) were used in the summer of 2004 to determine the origins of fecal pollution at the two beaches. Both MST methods detected substantial human-origin pollution at the two beaches, in locations producing consistently high levels of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of the mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE).
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Playas/normas , Enterococcus/aislamiento & purificación , Restauración y Remediación Ambiental/métodos , Heces/microbiología , Aguas del Alcantarillado/microbiología , Farmacorresistencia Microbiana , Electroforesis en Gel de Campo Pulsado , Fluorometría , Humanos , Especificidad de la Especie , VirginiaRESUMEN
Antibiotic resistance analysis (ARA), frequency of sampling, and seasonality were evaluated in a rural Virginia watershed dominated by cattle. The selected watershed (Mill Creek) was 3767 ha in size, included two small communities (one sewered and one unsewered), and several farms that when combined contained over 3800 beef and dairy cattle. Monthly monitoring of fecal coliforms at two sampling sites in Mill Creek from January to December, 2001, revealed that the recreational standard (1000 colony forming units, CFUs/100 ml) was exceeded a total of eight times for a 33% violation rate at each site. In addition, stream samples were collected weekly for 4 consecutive weeks during seasonal high flows (March) and seasonal low flows (September-October), plus daily for 7 consecutive days within the weekly schedules for a combined total of 60 stream samples (30 at each of two sites). The recreational standard was exceeded once during seasonal high flow and nine times during seasonal low flow. Microbial source tracking (MST) was performed by ARA to assess the impact of cattle on water quality within the different sampling routines. The resistance patterns of 2880 water isolates and 1158 known source (host-origin) isolates were determined with seven antibiotics at 28 different concentrations. The 1158 isolate database was reduced to 562 unique isolates when clonal ARA patterns were removed. This database of 562 unique isolates had an average rate of correct classification (ARCC) of 95.4%, and several statistical procedures confirmed the library as accurate and representative. Sixty-five percent of 50 challenge-set isolates from sources, but not samples, used in the library were correctly identified. The 562 unique pattern database was used to classify Escherichia coli isolates from water samples into six host source categories. The ARA results showed that cattle were the major source of pollution in the stream and cattle were the dominant source in over 60% of the water samples. Sampling frequency and seasonality had no effect on the MST results, as cattle dominated both seasons and samplings. Deer were a minor contributor in the summer (high water demand), and geese were a minor contributor in the winter when migratory flocks were observed moving through the watershed. An unexpected human allocation was found, especially under seasonal high flow conditions. The exact origin of this human allocation is not known. This project demonstrated that a host-origin library, based on a phenotypic method, could be developed for a well-defined watershed and was both representative of the sources in the watershed and performed reasonably well against a challenge set.
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Bacterias/aislamiento & purificación , Farmacorresistencia Microbiana , Agua Dulce/microbiología , Contaminación del Agua/análisis , Animales , Animales Domésticos , Bovinos , Salud Rural , Estaciones del Año , Aguas del Alcantarillado/microbiología , Virginia , Contaminantes del Agua/análisisRESUMEN
A new library-based microbial source tracking (MST) approach intended for initial application in the coastal waters of Virginia was evaluated. Host-origin isolates of Enterococcus spp. were collected from beaches and the surrounding tidewater region of Virginia and used to construct a library based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer (IGS) region, and subsequent digestion with MboI. Initial results from small host-origin libraries (64 and 200 total isolates) with discriminant analysis (DA) and logistic regression (LR) yielded high average rates of correct classification (ARCC) for a four-source classification split (birds, dogs, sewage, and wildlife), with ARCCs ranging from 83 to 100%. However, the poor results obtained when classification was attempted on a non-library validation set (VS, ARCCs of 47 and 48%, respectively, using DA and LR) demonstrated that a library of 200 isolates was insufficient to adequately represent the diversity of the enterococci in the sampled region. An increase in the library size to 1029 total isolates was accompanied by a reduction in the ARCC of the library to 42.7% with DA and 45.7% with LR, plus similarly poor results obtained from the VS. The low correct classification rates generated by the larger known-source library were unsuitable for field application. Many reported MST methods have been based on results obtained using small host-origin libraries without external validation. Our results indicate that such an approach can be very misleading, and that larger libraries and external validation is essential for the confirmation of preliminary results.
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ADN Ribosómico/análisis , ADN Ribosómico/genética , Enterococcus/genética , Animales , ADN Ribosómico/clasificación , Enterococcus/clasificación , Enterococcus/aislamiento & purificación , Heces/microbiologíaRESUMEN
These studies indicate that the interconversions of delta 1-pyrroline-5-carboxylate and proline can function as a shuttle that generates extra-mitochondrial NADP+ and transfers hydride ions into mitochondria in a cell-free rat liver system. A phosphate-free buffer with high concentrations of triethanolamine and 2-mercaptoethanol prevented the cold inactivation of pyrroline-5-carboxylate reductase (EC 1.5.1.2) in liver extracts. This enzyme had an apparent KmNADPH that was 2% of the apparent KmNADH X VmaxNADPH was approx. 50% of VmaxNADH. Unlabeled proline was converted to [5-3H]proline in incubations containing liver soluble fraction, mitochondria and a [4S-3H]NADPH generating system. This demonstrated one turn of the proposed shuttle in a homologous liver system. [5-3H]Proline production increased linearly over 60 min and decreased by 87% or more when specific components were eliminated. Rotenone was required for maximal activity, suggesting that inhibition of delta 1-pyrroline-5-carboxylate efflux would be required for significant shuttle activity in vivo. Both the relative concentrations of NADPH and NADH in liver cytosol and the kinetic characteristics of liver pyrroline-5-carboxylate reductase predict that the described shuttle should be overwhelmingly linked to NADPH rather than NADH. A NADPH-linked delta 1-pyrroline-5-carboxylate-proline shuttle may occur in hepatocytes and function at specific times to regulate pathways limited by cytosolic [NADP+].
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Mitocondrias Hepáticas/metabolismo , NADP/metabolismo , Prolina/metabolismo , Pirroles/metabolismo , Animales , Sistema Libre de Células , Frío , Oxidación-Reducción , Vía de Pentosa Fosfato , Pirrolina Carboxilato Reductasas/metabolismo , Ratas , SolubilidadRESUMEN
To explore the mechanism of erythropoietin action on differentiation of erythroblasts, we have examined its effect on regulating phosphorylation of the 25-kD mRNA cap binding protein (eIF-4E). Erythroblasts from the spleens of mice infected with the anemia strain of Friend virus (FVA cells) were studied. Erythropoietin stimulated phosphorylation of eIF-4E in FVA cells within 30 minutes, and this effect was maximal at 60 minutes. Phosphoamino acid analysis and tryptic phosphopeptide map analysis of eIF-4E isolated from both control and erythropoietin-treated cells identified a predominant phosphopeptide containing phosphoserine. However, when cells were incubated with 1 muM okadaic acid, eIF-4E was phosphorylated on both serine and threonine residues and three additional tryptic phosphopeptides were detected. We also identified a 37-kD phosphoprotein (pp37) that bound specifically to the m7GTP cap structure and coimmunoprecipitated with eIF-kD protein was phosphorylated on both serine and threonine residues. These results indicate that phosphorylation of eIF-4E is a target in erythropoietin-initiated signal transduction events and that this phosphorylation precedes observable effects of erythropoietin on macromolecular biosynthesis. Although of pp37 remains to be studied, it may represent a developmentally regulated mRNA cap binding protein.
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Eritroblastos/metabolismo , Eritropoyetina/farmacología , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Anemia/metabolismo , Anemia/virología , Animales , Éteres Cíclicos/farmacología , Factor 4E Eucariótico de Iniciación , Femenino , Virus de la Leucemia Murina de Friend , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Ratones , Peso Molecular , Ácido Ocadaico , Fosforilación , Bazo/citología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Binding of eIF-4E to the 5' m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gamma-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein eIF-4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [gamma-32P]8-N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)-chelate chromatography and reverse-phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled eIF-4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122.Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. A detailed understanding of eIF-4E.m7G mRNA cap interactions may lead the way to regulating this essential protein-RNA interaction for specific mRNA in vivo.
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Factores de Iniciación de Péptidos/química , ARN Mensajero/metabolismo , Marcadores de Afinidad , Secuencia de Aminoácidos , Factor 4E Eucariótico de Iniciación , Humanos , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
cDNA sequence studies have predicted the existence of protein kinases that have not been characterized at the protein level. This laboratory has previously shown that polyclonal rabbit antibodies directed against a highly conserved sequence motif, GTPEYLAPE, present in the catalytic domain of many protein kinases will react during immunoblots with several protein kinases containing the same or homologous motifs. In this report I describe the development of a mouse monoclonal antibody, designated APE-1, that reacts with the GTPEYLAPE motif. In addition, the use of this monoclonal antibody in the affinity purification of kinase activities from rabbit reticulocyte lysate is described. This approach may provide a general method for the purification of previously uncharacterized protein kinases that share regions which are homologous to the GTPEYLAPE motif. Refinements in this method may permit the rapid purification of nonabundant and/or rapidly inactivated protein kinases that have not been isolated using other approaches.
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Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Proteínas Quinasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Indicadores y Reactivos , Ratones , Datos de Secuencia Molecular , Proteínas Quinasas/química , Proteínas Quinasas/inmunología , Conejos , Reticulocitos/enzimologíaRESUMEN
Platelet-derived growth factor exerts rapid effects on protein synthesis and polysome formation in cultured cells. We report that platelet-derived growth factor stimulates a rapid phosphorylation of eIF-4E in WI-38 human lung fibroblasts. The effect was dependent on both time and PDGF concentrations. Phosphoserine was the sole phosphoamino acid identified and tryptic phosphopeptide maps showed a single phosphopeptide under both control and PDGF conditions. Phosphorylation of eIF-4E may be of the events required for initiating entry into G1 and commitment into S phase of the cell cycle.
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Proteínas Portadoras/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Aminoácidos/análisis , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Factor 4E Eucariótico de Iniciación , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Cinética , Pulmón , Peso Molecular , Factores de Iniciación de Péptidos/aislamiento & purificación , Mapeo Peptídico , Fosfopéptidos/aislamiento & purificación , Fosforilación , Proteínas de Unión a Caperuzas de ARNRESUMEN
The phosphorylation and dephosphorylation of the 25 kDa mRNA cap binding protein eukaryotic initiation factor-4E (eIF-4E) is regulated during different physiologic and pathophysiologic states that include cell growth and the late phase of adenovirus infection. We have found that okadaic acid is much more effective in increasing the phosphorylated fraction of eIF-4E than phorbol 12-myristate 13-acetate in Hep G2 cells. Phosphoprotein phosphatase 2A dephosphorylated eIF-4E isolated from both phorbol 12-myristate 13-acetate- or okadaic acid-treated cells, whereas alkaline and acid phosphatase were relatively ineffective. The ability of purified [35S]eIF-4E isolated from okadaic acid-treated cells to bind mRNA caps was compared to phosphoprotein phosphatase 2A-treated [35S]eIF-4E and found to be no different. This suggests that alternative explanations for the previously observed effects of eIF-4E phosphorylation on protein synthesis must be considered. In addition, our results indicate that the in vivo phosphorylation of eIF-4E is not catalyzed solely by protein kinase C.
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Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Caperuzas de ARN/metabolismo , Éteres Cíclicos/farmacología , Factor 4E Eucariótico de Iniciación , Humanos , Hígado/enzimología , Modelos Biológicos , Ácido Ocadaico , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2 , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Using a synthetic oligopeptide (CGGGTPEYLAPEGGK) crosslinked to keyhole limpet hemocyanin we have raised polyclonal rabbit antibodies against a 9 residue homologous region found in the catalytic domain of most protein kinases. These antibodies reacted during Western immunoblotting with cAMP dependent protein kinase catalytic subunit, phosphorylase kinase gamma subunit and calcium calmodulin dependent protein kinase II which have homologous sequences of GTPEYLAPE, GTPSYLAPE and GTPGYLSPE, respectively. Five other protein kinases did not react with anti-GTPEYLAPE antibodies during Western immunoblotting. Affinity-purified antibodies were able to detect as little as 50 ng of cAMP dependent protein kinase and 200 ng of Ca2+/calmodulin dependent protein kinase II. Immunoblotting of A431 cell plasma membrane vesicles indicated the presence of an approximately 55 kDa protein that contains the conserved sequence and is likely to be a protein kinase. Antibodies directed against conserved sequences present in protein kinases, or possibly other enzymes, may be useful in identifying previously uncharacterized enzymes at the protein level.
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Anticuerpos , Oligopéptidos/síntesis química , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Anticuerpos/aislamiento & purificación , Sitios de Unión , Western Blotting , Línea Celular , Membrana Celular/enzimología , Epítopos/análisis , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas Quinasas/inmunología , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Addition of L-pyrroline-5-carboxylic acid to reticulocyte lysates inhibits protein synthesis and induced phosphoproteins of 25 and 14 kDa. The 25 kDa phosphoprotein had the same Mr and pI as phosphorylated eIF-4E. Incubation of lysates with L-pyrroline-5-carboxylic acid did not alter the crosslinking of eIF-4E to reovirus mRNA caps. These results suggest that modifications of the translational apparatus other than eIF-4E phosphorylation may mediate the inhibitory effect seen with L-pyrroline-5-carboxylic acid and/or that phosphorylation of eIF-4E may effect functions subsequent to its interaction with the mRNA cap such as protein-protein interactions with other cap-specific translation factors.
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Factores de Iniciación de Péptidos/metabolismo , Inhibidores de la Síntesis de la Proteína , Pirroles/farmacología , Animales , Factor 4E Eucariótico de Iniciación , Técnicas In Vitro , Punto Isoeléctrico , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilación , Caperuzas de ARN/metabolismo , Conejos , ReticulocitosRESUMEN
The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients. Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication. In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity. This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template. The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30 degrees C. Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme. Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors.
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Escherichia coli/metabolismo , Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/biosíntesis , Proteínas no Estructurales Virales/biosíntesis , Expresión Génica , Globinas/genética , Hepacivirus/genética , Humanos , Mutagénesis , Poli U/metabolismo , ARN Mensajero/metabolismo , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Moldes Genéticos , Proteínas no Estructurales Virales/genéticaRESUMEN
Pyrroline-5-carboxylate is the intermediate in the interconversions of proline, ornithine, and glutamate. Recent studies have suggested that pyrroline-5-carboxylate has regulatory properties in a wide range of tissues. Physiologic studies in humans have been limited by the unavailability of a sufficiently sensitive assay for this compound. Until now pyrroline-5-carboxylate has not been detectable in plasma of normal humans or even in plasma of patients with type II hyperprolinemia. We now report a method for measuring pyrroline-5-carboxylate made possible by a preparation of purified Escherichia coli pyrroline-5-carboxylate reductase. This method is more sensitive than currently available methods by two or three logs and is applicable for all biologic fluids. We have quantitated pyrroline-5-carboxylate in normal plasma, urine, and saliva. In addition, we have found pyrroline-5-carboxylate levels 10x to 20x normal in two patients with type II hyperprolinemia. The ability to measure physiologic concentrations of pyrroline-5-carboxylate now enables further characterization of its role as a regulatory molecule.