RESUMEN
RESULTS: Findings show how our respondents experience a high burden of disease (breathlessness, fatigue, exacerbations, loss of family, friends and employment) and treatment (oral corticosteroids' side-effects, dependency, life-style changes). Treatment with biologicals is relatively new for respondents. They mention to be cautious in their embrace of biologicals and in expressing hope for the future. Respondents who react to treatment with biologicals experience relief of both the burden of disease and treatment. They aim to regain their social life and societal participation, a contrast to those for whom biologicals prove ineffective. Biologicals' burden of treatment is experienced as low and minor side-effects are mentioned by three respondents. Respondents appear relatively unconcerned about the lack of knowledge concerning the long-term effects of biologicals.Conclusions: Effective treatment with biologicals is generally experienced as a cautiously optimistic next step in a much longer and complex process of living with severe asthma. The practical lessons we draw point to managing patients' expectations and the need to pay attention to patients not eligible for treatment with biologicals.
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Asma , Productos Biológicos , Corticoesteroides , Asma/terapia , Productos Biológicos/efectos adversos , Empleo , Humanos , Resultado del TratamientoRESUMEN
Soil organic carbon (SOC) regulates terrestrial ecosystem functioning, provides diverse energy sources for soil microorganisms, governs soil structure, and regulates the availability of organically bound nutrients. Investigators in increasingly diverse disciplines recognize how quantifying SOC attributes can provide insight about ecological states and processes. Today, multiple research networks collect and provide SOC data, and robust, new technologies are available for managing, sharing, and analyzing large data sets. We advocate that the scientific community capitalize on these developments to augment SOC data sets via standardized protocols. We describe why such efforts are important and the breadth of disciplines for which it will be helpful, and outline a tiered approach for standardized sampling of SOC and ancillary variables that ranges from simple to more complex. We target scientists ranging from those with little to no background in soil science to those with more soil-related expertise, and offer examples of the ways in which the resulting data can be organized, shared, and discoverable.
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Carbono , Suelo , Secuestro de Carbono , Ecosistema , NutrientesRESUMEN
As experts-by-experience, clients are thought to give specific input for and legitimacy to regulatory work. In this paper we track a 2017 pilot by the Dutch Health and Youth Care Inspectorate that aimed to use experiential knowledge in risk regulation through engaging with clients of long-term elderly care homes. Through an ethnographic inquiry we evaluate the design of this pilot. We find how the pilot transforms selected clients into experts-by-experience through training and site visits. In this transformation, clients attempt, and fail, to bring to the fore their definitions of quality and safety, negating their potentially specific contributions. Paradoxically, in their attempts to expose valid new knowledge on the quality of care, the pilot constructs the experts-by-experience in such a way that this knowledge is unlikely to be opened up. Concurrently, we find that in their attempts to have their input seen as valid, experts-by-experience downplay the value of their experiential knowledge. Thus, we show how dominating, legitimated interpretations of (knowledge about) quality of care resonate in experimental regulatory practices that explicitly try to move beyond them, emphasizing the need for a pragmatic and reflexive engagement with clients in the supervision of long-term elderly care.
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Hogares para Ancianos/normas , Cuidados a Largo Plazo/normas , Participación del Paciente/métodos , Calidad de la Atención de Salud/normas , Anciano , Femenino , Humanos , Masculino , Países Bajos , Participación del Paciente/psicología , Proyectos PilotoRESUMEN
Source-separated black water (BW) (toilet water) containing 38% of the organic material and 68% of the phosphorus in the total household waste (water) stream including kitchen waste, is a potential source for energy and phosphorus recovery. The energy recovered, in the form of electricity and heat, is more than sufficient for anaerobic treatment, nitrogen removal and phosphorus recovery. The phosphorus balance of an upflow anaerobic sludge blanket reactor treating concentrated BW showed a phosphorus conservation of 61% in the anaerobic effluent. Precipitation of phosphate as struvite from this stream resulted in a recovery of 0.22 kgP/p/y, representing 10% of the artificial phosphorus fertiliser production in the world. The remaining part of the phosphorus ended up in the anaerobic sludge, mainly due to precipitation (39%). Low dilution and a high pH favour the accumulation of phosphorus in the anaerobic sludge and this sludge could be used as a phosphorus-enriched organic fertiliser, provided that it is safe regarding heavy metals, pathogens and micro-pollutants.
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Fósforo/química , Aguas del Alcantarillado/análisis , Anaerobiosis , Reactores Biológicos , Precipitación Química , Conservación de los Recursos Energéticos , Compuestos de Magnesio/química , Fosfatos/química , EstruvitaRESUMEN
Vacuum collected black (toilet) water contains hormones and pharmaceuticals in relatively high concentrations (µg/L to mg/L range) and separate specific treatment has the potential of minimizing their discharge to surface waters. In this study, the fate of estrogens (natural and synthetical hormones) and pharmaceuticals (paracetamol, metoprolol, propranolol, cetirizine, doxycycline, tetracycline, ciprofloxacin, trimethoprim, carbamazepine, ibuprofen and diclofenac) in the anaerobic treatment of vacuum collected black water followed by nitrogen removal by partial nitritation-anammox was investigated. A new analytical method was developed to detect the presence of several compounds in the complex matrix of concentrated black water. Detected concentrations in black water ranged from 1.1 µg/L for carbamazepine to >1000 µg/L for paracetamol. Anaerobic treatment was only suitable to remove the majority of paracetamol (>90%). Metoprolol was partly removed (67%) during aerobic treatment. Deconjugation could have affected the removal efficiency of ibuprofen as concentrations even increased during anaerobic treatment and only after the anammox treatment 77% of ibuprofen was removed. The presence of persistent micro-pollutants (diclofenac, carbamazepine and cetirizine), which are not susceptible for biodegradation, makes the application of advanced physical and chemical treatment unavoidable.
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Nitrógeno/aislamiento & purificación , Eliminación de Residuos Líquidos/métodos , Purificación del Agua/métodos , Anaerobiosis , HormonasRESUMEN
Black (toilet) water contains half of the organic load in the domestic wastewater, as well as the major fraction of the nutrients nitrogen and phosphorus. When collected with vacuum toilets, the black water is 25 times more concentrated than the total domestic wastewater stream, i.e. including grey water produced by laundry, showers etc. A two-stage nitritation-anammox process was successfully employed and removed 85%-89% of total nitrogen in anaerobically treated black water. The (free) calcium concentration in black water was too low (42 mg/L) to obtain sufficient granulation of anammox biomass. The granulation and retention of the biomass was improved considerably by the addition of 39 mg/L of extra calcium. This resulted in a volumetric nitrogen removal rate of 0.5 gN/L/d, irrespective of the two temperatures of 35 °C and 25 °C at which the anammox reactors were operated. Nitrous oxide, a very strong global warming gas, was produced in situations of an incomplete anammox conversion accompanied by elevated levels of nitrite.
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Calcio/química , Nitrógeno/aislamiento & purificación , Eliminación de Residuos Líquidos/métodos , Procesos Autotróficos , Nitrógeno/químicaRESUMEN
BACKGROUND: To study current clinical practice in blood glucose (BG) control in adult intensive care units (ICUs) in the Netherlands. METHODS: We performed a national survey focusing on blood glucose targets, insulin administration, BG control guidelines, and opinions regarding BG control aiming for normoglycaemia (known as intensive insulin therapy, IIT). RESULTS: The completed questionnaire was returned by 88/113 (78%) of the participating centres. In 98% (86/88) of the ICUs some sort of BG control was being practised. Half of the ICUs (42/86, 48%) used tight BG targets as with IIT; 28/86 (33%) and 13/86 (15%) used more liberal targets of 4.4 to 7.0 mmol/l and 4.4 to 8.0 mmol/l, respectively. Eighty-two (93%) reported having a local guideline on BG control (or IIT). The BG threshold to start insulin was 7.0+/-1.3 mmol/l vs 7.8+/-1.3 mmol/l in ICUs that practised IIT vs ICUs that practised less tight BG control, respectively (p=0.005). In 28/86 (33%) measurement of the BG values was done according to a strict time schedule (i.e., BG values were measured on predefined time points). While respondents were fairly agreed on the benefits of IIT, opinions regarding ease of implementation and time needed to apply this strategy varied. In addition, severe hypoglycaemia was considered a serious side effect of IIT. CONCLUSION: Approximately half of the ICUs in the Netherlands reported having implemented IIT. However, the full guideline as used in the original studies on IIT was hardly ever implemented. Concerns about severe hypoglycaemia, at least in part, hampers implementation of IIT.
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Glucemia , Enfermedad Crítica , Hiperglucemia/prevención & control , Unidades de Cuidados Intensivos , Adulto , Automonitorización de la Glucosa Sanguínea , Intervalos de Confianza , Encuestas de Atención de la Salud , Humanos , Hipoglucemia/etiología , Hipoglucemia/prevención & control , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/uso terapéutico , Insulina/efectos adversos , Insulina/uso terapéutico , Modelos Logísticos , Análisis Multivariante , Países Bajos , Oportunidad Relativa , Guías de Práctica Clínica como Asunto , Encuestas y CuestionariosRESUMEN
Black water (toilet water) contains half the load of organic material and the major fraction of the nutrients nitrogen and phosphorus in a household and is 25 times more concentrated, when collected with a vacuum toilet, than the total wastewater stream from a Dutch household. This research focuses on the partial nitritation of anaerobically treated black water to produce an effluent suitable to feed to the anammox process. Successful partial nitritation was achieved at 34 degrees C and 25 degrees C and for a long period (almost 400 days in the second period at 25 degrees C) without strict process control a stable effluent at a ratio of 1.3 NO(2)-N/NH(4)-N was produced which is suitable to feed to the anammox process. Nitrite oxidizers were successfully outcompeted due to inhibition by free ammonia and nitrous acid and due to fluctuating conditions in SRT (1.0-17 days) and pH (from 6.3 to 7.7) in the reactor. Microbial analysis of the sludge confirmed the presence of mainly ammonium oxidizers. The emission of nitrous oxide (N(2)O) is of growing concern and it corresponded to 0.6-2.6% (average 1.9%) of the total nitrogen load.
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Óxido Nitroso/análisis , Compuestos Orgánicos/análisis , Aguas del Alcantarillado/química , Eliminación de Residuos Líquidos/métodos , Algoritmos , Anaerobiosis , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Biodegradación Ambiental , Biomasa , Reactores Biológicos , Concentración de Iones de Hidrógeno , Hibridación Fluorescente in Situ , Nitritos/análisis , Nitritos/metabolismo , Óxido Nitroso/metabolismo , Compuestos Orgánicos/metabolismo , Compuestos de Amonio Cuaternario/análisis , Compuestos de Amonio Cuaternario/metabolismo , ARN Ribosómico 16S/genética , Aguas del Alcantarillado/microbiología , Factores de TiempoRESUMEN
The mechanics of microtubules, cylindrical protein filaments that constitute the cytoskeleton, have been well characterized on long length scales. Here, we investigate the persistence length of short (approximately 0.1 microm) ends of microtubules by measuring the trajectories of kinesin-propelled microtubules under perpendicular electric forces. We relate the measured trajectory curvatures to the biased thermal fluctuations of the leading microtubule end, and upon including all electrohydrodynamic forces, we find that the persistence length of the microtubule ends is only 0.08 +/- 0.02 mm. This is significantly shorter than the well established value of approximately 4-8 mm that is measured for long microtubules. Our data are in good agreement with recent theoretical predictions that microtubules mechanically behave as a loose assembly of independent protofilaments on these short length scales.
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Microtúbulos/química , Animales , Fenómenos Biomecánicos , Bovinos , Drosophila melanogaster , ElectricidadRESUMEN
We use micrometer-sized fluidic channels to confine and measure electrophoresis of freely suspended individual microtubules. We measure orientation-dependent velocities of microtubules and the electro-osmotic flow mobility in our channels to infer the anisotropic electrophoretic mobility of microtubules under physiological conditions. We discuss the difference between electrophoresis and purely hydrodynamic motion and its implications for interpreting mobility measurements. We show that the mobility anisotropy is a factor of 0.83, clearly different from the well known anisotropy factor of 0.5 in Stokes drag coefficients for cylindrical objects. We also show that the velocity is independent of microtubule length, which would be different for hydrodynamic motion. We demonstrate that the electric force on the counterions has important consequences for the interpretation of electrophoresis experiments and that ignoring this can lead to an underestimation of the effective charge by orders of magnitude. From the electrophoresis measurements, we calculate an effective surface-charge density of -36.7 +/- 0.4 mC/m2 for microtubules. Electrophoretic measurements of subtilisin-digested microtubules, which have the negatively charged C termini on the outer surface removed, show a 24% decrease in mobility and, correspondingly, in surface charge, but no change in anisotropy.
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Microtúbulos/fisiología , Anisotropía , Dimerización , Electroforesis , Microtúbulos/química , Soluciones , Propiedades de Superficie , Tubulina (Proteína)/químicaRESUMEN
The number of indications for the medical use of melatonin is slowly increasing. Melatonin is produced by the pineal gland and is a key signal in the circadian rhythm of the body. Melatonin plays an obvious role in the pathophysiology and treatment of sleep disorders and jetlag. Recent research has also demonstrated its favourable effect on blood-pressure regulation. By analogy, melatonin may play a role in a variety of other circadian processes. However, research into the precise effects is still insufficient.
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Ritmo Circadiano/fisiología , Melatonina/fisiología , Melatonina/uso terapéutico , Glándula Pineal/fisiología , Presión Sanguínea/fisiología , Humanos , Trastornos del Sueño-Vigilia/tratamiento farmacológico , Trastornos del Sueño-Vigilia/etiología , ViajeRESUMEN
To examine the bioavailability of rifampicin formulations produced in Indonesia, we conducted a single-dose, double-blind, cross-over bioavailability study. Antituberculosis drugs from three Indonesian manufacturers and one international manufacturer were compared in 12 healthy Indonesian subjects. Out of three local manufacturers, two showed equal bioavailability compared to the reference standard, and one showed slightly lower bioavailability (ratio 0.86; 90% confidence interval 0.80-0.91) and substandard rifampicin content of drug preparations. Plasma rifampicin concentrations in this study were more than three-fold higher than concentrations recently found in tuberculosis patients in Indonesia, which suggests that unknown (disease-related) determinants may reduce the bioavailability of rifampicin formulations.
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Antituberculosos/farmacocinética , Rifampin/farmacocinética , Adulto , Disponibilidad Biológica , Estudios Cruzados , Método Doble Ciego , Humanos , Indonesia , Masculino , Valores de ReferenciaRESUMEN
Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural minus-strand RNAs (containing any posttranscriptional modification that might have occurred) and have tested them for coat protein binding sites and template activity in an in vitro system with the viral RNA polymerase. The enzyme was prepared by an advanced isolation procedure. All three minus strands had a single non-coded G at their 3' terminus. They were not able to withdraw coat protein subunits from virions as free virion RNAs do. No sites protected by coat protein against ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters. Except for minus RNA 3 which caused a minute amount of full-size plus strand to be synthesized, the minus strands did not function as templates for full-size complementary strands. On the other hand, they gave rise to a number of well-defined shorter products, the synthesis of which was stimulated by the addition of coat protein. These products could not be elongated by a chase treatment and were probably the result of internal initiations. It is concluded that, although posttranscriptional modifications of the template and the presence of coat protein may be necessary factors for plus-strand RNA synthesis, they are certainly not sufficient. Our purified in vitro system needs further sophistication.
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Virus del Mosaico de la Alfalfa/fisiología , Cápside/metabolismo , ARN Polimerasas Dirigidas por ADN/farmacología , ARN Viral/metabolismo , Regiones no Traducidas 3'/genética , Virus del Mosaico de la Alfalfa/enzimología , Virus del Mosaico de la Alfalfa/genética , Sitios de Unión , Guanosina/genética , Técnicas In Vitro , Unión Proteica , ARN Viral/biosíntesis , Moldes Genéticos , Transcripción Genética , Replicación Viral/genéticaRESUMEN
The presence of the HIV-protease inhibitor indinavir in saliva was analyzed to investigate whether salivary indinavir concentrations are applicable to monitor compliance and/or predict plasma indinavir levels. Fourteen HIV-infected outpatients treated with indinavir and 24 healthy volunteers who ingested a single dose of indinavir were included. Paired plasma and citric-acid-stimulated saliva samples were analyzed by high-performance liquid chromatography (HPLC). Stimulated salivary indinavir concentrations showed a high correlation (r = 0.85, p < 0.01) with corresponding plasma levels. The median saliva/plasma ratio was 65% (P25 50%; P75 94%). The ratios were independent of the plasma concentration; however, a relation with time after ingestion was seen. The unbound fraction of indinavir in plasma was not significantly correlated with the saliva/plasma ratio after stimulated saliva collection, in contrast with a subset of nonstimulated saliva from healthy volunteers, where we did find a significant correlation. Although stimulated salivary indinavir concentrations are highly correlated with plasma concentrations, it is not possible to predict plasma indinavir levels by the salivary concentrations for purposes of therapeutic drug monitoring, due to large interindividual and intraindividual variation. Nevertheless, monitoring compliance by measuring the presence of indinavir in saliva is possible: ingestion of indinavir can be assessed with a sensitivity of 84.8% in the whole dosing interval or with 98.8% between 1 and 6 hours after the last dose, which is comparable with plasma.
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Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/farmacocinética , Indinavir/farmacocinética , Cooperación del Paciente , Saliva/metabolismo , Adolescente , Adulto , Femenino , Infecciones por VIH/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Masculino , Persona de Mediana Edad , Orosomucoide/análisisRESUMEN
We have designed and used an in vitro bone marrow cell culturing system for investigating pharmacodynamic interactions between platinum anti-cancer drugs and taxanes. With this system, in which the bone marrow progenitor cell CFU-GM is proliferating and differentiating into granulocytes and monocytes, we could show a strong antagonistic cytotoxicity of the combination carboplatin and Taxotere, in three different schedules, and of the combination cisplatin and Taxol, in two out of the three schedules tested. Modulation of intracellular platinum drug accumulation in granulocytes and monocytes does not seem to be a plausible explanation for the observed antagonism. In vitro co-incubation of granulocytes/monocytes with the combination carboplatin and Taxotere did not reveal an effect of Taxotere on intracellular platinum accumulation. Although Taxol reduced intracellular cisplatin levels by 12%, this effect was not significantly different from the co-incubation of cisplatin with Cremophor EL, the solvent for paclitaxel in Taxol. The toxicity data obtained in this study seem to be in accordance with recent clinical trials where combination therapies with platinum drugs and taxanes resulted in marked reductions in myelosuppression in patients. Therefore, these types of assays could be useful as to the assessment of bone marrow toxicities of clinically important drug combinations.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Células de la Médula Ósea/efectos de los fármacos , Carboplatino/administración & dosificación , Cisplatino/administración & dosificación , Paclitaxel/análogos & derivados , Paclitaxel/administración & dosificación , Taxoides , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Células de la Médula Ósea/metabolismo , Células Cultivadas/efectos de los fármacos , Docetaxel , Antagonismo de Drogas , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Polisorbatos/administración & dosificaciónRESUMEN
As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.
Asunto(s)
ARN Polimerasa II/química , Baculoviridae , Clonación Molecular , Cisteína/análisis , Glutatión/metabolismo , Humanos , Metionina/análisis , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , ARN Polimerasa II/genéticaRESUMEN
A sensitive high-perforrmance liquid chromatographic assay has been developed to determine the concentrations of the HIV-protease inhibitor indinavir in human plasma. The sample pretreatment involved a protein precipitation procedure using 100 microl of human plasma and 400 microl of acetonitrile. Chromatography was carried out on an Octadecyl column using a mobile phase of acetonitrile-water (40:60, v/v). The water phase contained 50 mM phosphate buffer pH 6 and 4 g/l tetramethylammoniumchloride. Ultraviolet detection at 210 nm was used. The method has been validated with regard to specificity, detection limit, lower and upper limit of quantitation, recovery, accuracy, and inter- and intra-assay precision. Stability tests under various conditions were performed. The bioanalytical assay is now in use for the determination of indinavir in several clinical pharmacokinetic studies in HIV-infected patients.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Infecciones por VIH/sangre , Inhibidores de la Proteasa del VIH/sangre , Indinavir/sangre , Acetonitrilos/química , Administración Oral , Adulto , Ritmo Circadiano , Estabilidad de Medicamentos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Inhibidores de la Proteasa del VIH/administración & dosificación , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacocinética , Humanos , Indinavir/administración & dosificación , Indinavir/química , Indinavir/farmacocinética , Masculino , Persona de Mediana Edad , Concentración Osmolar , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrofotometría Ultravioleta , Factores de TiempoRESUMEN
In the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependent in vitro RNA synthesizing system of alfalfa mosaic virus (AIMV) we were able to study the effect of small chemical modifications of the 3' end of the template RNAs on product formation. After oxidation of the 3'-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuyl et al., Virology 176, 346-354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3' end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AIMV displays a high specificity for its cognate template, either before or after the oxidation of the 3'-terminal nucleoside. From this it was concluded that periodate modification of the 3'-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadt et al., Virology 182, 309-315, 1991), was not the main target involved in the inhibition of RNA synthesis.
Asunto(s)
Virus del Mosaico de la Alfalfa/metabolismo , Proteínas de la Cápside , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Virus del Mosaico de la Alfalfa/genética , Compuestos de Anilina/farmacología , Cápside/metabolismo , Radical Hidroxilo , Nucleótidos/metabolismo , Oxidación-Reducción , Ácido Peryódico/farmacología , Monoéster Fosfórico Hidrolasas/metabolismo , ARN Viral/metabolismo , Moldes GenéticosRESUMEN
An RNA-dependent RNA polymerase (RdRp) purified from alfalfa mosaic virus-infected tobacco is capable of synthesizing in vitro full-size RNAs of minus and plus polarities. However, the enzyme is not able to perform a complete replication cycle in vitro. The products were found to be completely base-paired to their templates. The enzyme was able to use double-stranded RNA as a template for RNA synthesis if it could initiate from a single-stranded promoter. The inability (of most) of our enzyme preparations to create a single-stranded initiation site could explain why they could not perform a complete replication cycle in vitro. This is the first report on duplex RNA unwinding activities by a plant viral RdRp.
Asunto(s)
Virus del Mosaico de la Alfalfa/enzimología , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , ARN Bicatenario/metabolismoRESUMEN
The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of cost protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated that viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E. M. Taschner et al., 1991, Virology 181, 687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and C-RdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minus-strand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfalfa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.