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BACKGROUND AND PURPOSE: To clarify the relevance of titres of IgG antibodies against contactin-associated protein-2 (CASPR2) in diagnosing anti-CASPR2 encephalitis and to describe features and outcomes. METHODS: This was a retrospective analysis of 64 patients with CASPR2 antibodies, categorized independently as 'autoimmune encephalitis' or 'other disease'. Logistic regression methods were performed to identify potential predictors of 'autoimmune encephalitis' in addition to CASPR2 antibodies. RESULTS: An upfront CASPR2 antibody serum titre cut-off at ≥1:200 had a diagnostic sensitivity of 85% and a specificity of 81%. Logistic regression analyses indicated that, in addition to titre, encephalitic magnetic resonance imaging (MRI) was a significant predictor of 'autoimmune encephalitis' (Nagelkerke's R2 = 0.81, P < 0.001) with high sensitivity (84%) and very high specificity (100%). Patients with CASPR2 antibodies and an estimated probability of >70% of having anti-CASPR2 encephalitis (n = 22) had limbic encephalitis (n = 18, one patient plus ataxia), Morvan syndrome (n = 2) or a hyperkinetic movement disorder (n = 2). Median modified Rankin score (mRS) at diagnosis was 3 (range 1-4). Twenty patients were male; median age was 64 (range 54-75) years; 5/15 patients with cerebrospinal fluid data had intrathecal CASPR2 antibody synthesis, and 12/19 with follow-ups >3 months (median 12 months, range 4-43 months) improved by ≥1 mRS point resulting in a median mRS of 2 (range 0-6; one death; all but one having received immunotherapy); and 2/15 patients with follow-up MRI developed hippocampal atrophy. CONCLUSIONS: Only higher CASPR2 serum antibody titres indicate anti-CASPR2 encephalitis, and diagnostic accuracy increases if MRI findings are considered. Anti-CASPR2 encephalitis has characteristic features and a favourable outcome with immunotherapy.
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Autoanticuerpos/sangre , Encefalitis/diagnóstico , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Anciano , Encefalitis/sangre , Encefalitis/diagnóstico por imagen , Encefalitis/inmunología , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Anaphylaxis caused by hymenoptera venom allergy is associated with elevation of baseline serum tryptase (sBT) and/or mastocytosis in about 5% of patients. Up to now, no information has become available on single venom allergen sIgE reactivity and the usefulness of component-resolved approaches to diagnose this high-risk patient group. To address the component-resolved sIgE sensitization pattern and diagnostic sensitivity in hymenoptera venom-allergic patients with elevated sBT levels and/or mastocytosis, a panel of yellow jacket and honeybee venom allergens was applied on a widely used IgE immunoassay platform. METHODS: Fifty-three patients with mastocytosis and/or elevated sBT tryptase level and systemic reactions to hymenoptera venoms were analyzed for their IgE reactivity to recombinant yellow jacket and honeybee venom allergens by Immulite3 g. RESULTS: sIgE reactivity to Ves v 1, Ves v 5, Api m 1 to Api m 4 and Api m 10 was found at a similar frequency in hymenoptera venom-allergic patients with and without elevated sBT levels and/or mastocytosis. However, the use of the recombinant allergens and a diagnostic cutoff of 0.1 kUA /L allowed the diagnosis of patients with otherwise undetectable IgE to venom extract. The diagnostic sensitivity of yellow jacket venom allergy using the combination of Ves v 1 and Ves v 5 was 100%. CONCLUSIONS: In high-risk patients with elevated sBT levels and/or mastocytosis, the use of molecular components and decreasing the threshold sIgE level to 0.1 kUA /L may be needed to avoid otherwise undetectable IgE to hymenoptera venom extracts in about 8% of such patients.
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Anafilaxia/sangre , Anafilaxia/diagnóstico , Anafilaxia/etiología , Venenos de Artrópodos/inmunología , Himenópteros/inmunología , Mastocitosis/sangre , Triptasas/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alérgenos/inmunología , Animales , Especificidad de Anticuerpos/inmunología , Biomarcadores , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Mastocitosis/diagnóstico , Persona de Mediana Edad , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
BACKGROUND: Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation. Furthermore, the presence of Api m 10 in honeybee venom (HBV) and licensed venom immunotherapy preparations was addressed. METHODS: Api m 10 was produced as soluble, aglycosylated protein in Escherichia coli and as differentially glycosylated protein providing a varying degree of fucosylation in insect cells. IgE reactivity and basophil activation of allergic patients were analyzed. For detection of Api m 10 in different venom preparations, a monoclonal human IgE antibody was generated. RESULTS: Both, the aglycosylated and the glycosylated variant of Api m 10 devoid of cross-reactive carbohydrate determinants (CCD), exhibited IgE reactivity with approximately 50% of HBV-sensitized patients. A corresponding reactivity could be documented for the activation of basophils. Although the detection of the native protein in crude HBV suggested content comparable to other relevant allergens, three therapeutical HBV extracts lacked detectable amounts of this component. CONCLUSION: Api m 10 is a genuine allergen of A. mellifera venom with IgE sensitizing potential in a significant fraction of allergic patients independent of CCD reactivity. Thus, Api m 10 could become a key element for component-resolved diagnostic tests and improved immunotherapeutic approaches in hymenoptera venom allergy.
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Alérgenos/inmunología , Venenos de Abeja/inmunología , Abejas/inmunología , Alérgenos/genética , Alérgenos/uso terapéutico , Animales , Basófilos/inmunología , Venenos de Abeja/genética , Venenos de Abeja/uso terapéutico , Abejas/genética , Reacciones Cruzadas/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Mordeduras y Picaduras de Insectos/inmunología , Mordeduras y Picaduras de Insectos/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
Future climate warming is expected to enhance plant growth in temperate ecosystems and to increase carbon sequestration. But although severe regional heatwaves may become more frequent in a changing climate, their impact on terrestrial carbon cycling is unclear. Here we report measurements of ecosystem carbon dioxide fluxes, remotely sensed radiation absorbed by plants, and country-level crop yields taken during the European heatwave in 2003. We use a terrestrial biosphere simulation model to assess continental-scale changes in primary productivity during 2003, and their consequences for the net carbon balance. We estimate a 30 per cent reduction in gross primary productivity over Europe, which resulted in a strong anomalous net source of carbon dioxide (0.5 Pg C yr(-1)) to the atmosphere and reversed the effect of four years of net ecosystem carbon sequestration. Our results suggest that productivity reduction in eastern and western Europe can be explained by rainfall deficit and extreme summer heat, respectively. We also find that ecosystem respiration decreased together with gross primary productivity, rather than accelerating with the temperature rise. Model results, corroborated by historical records of crop yields, suggest that such a reduction in Europe's primary productivity is unprecedented during the last century. An increase in future drought events could turn temperate ecosystems into carbon sources, contributing to positive carbon-climate feedbacks already anticipated in the tropics and at high latitudes.
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Dióxido de Carbono/metabolismo , Productos Agrícolas/metabolismo , Desastres , Ecosistema , Efecto Invernadero , Calor , Atmósfera/química , Carbono/metabolismo , Europa (Continente) , Lluvia , Factores de TiempoRESUMEN
A fundamental question about human memory is which brain structures are involved, and when, in transforming experiences into memories. This experiment sought to identify neural correlates of memory formation with the use of intracerebral electrodes implanted in the brains of patients with temporal lobe epilepsy. Event-related potentials (ERPs) were recorded directly from the medial temporal lobe (MTL) as the patients studied single words. ERPs elicited by words subsequently recalled in a memory test were contrasted with ERPs elicited by unrecalled words. Memory formation was associated with distinct but interrelated ERP differences within the rhinal cortex and the hippocampus, which arose after about 300 and 500 milliseconds, respectively. These findings suggest that declarative memory formation is dissociable into subprocesses and sequentially organized within the MTL.
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Hipocampo/fisiología , Memoria/fisiología , Recuerdo Mental/fisiología , Lóbulo Temporal/fisiología , Adulto , Análisis de Varianza , Mapeo Encefálico , Electrodos Implantados , Epilepsia del Lóbulo Temporal/fisiopatología , Potenciales Evocados , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neuronas/fisiología , Factores de TiempoRESUMEN
BACKGROUND: An ethylacetate-soluble fraction (ET4) from the lichen Ramalina farinacea has previously been shown to inhibit the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. We now determined the antiviral activity of ET4 against other wild-type viruses, including the herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV). METHODS: Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of ET4 for 30 min at 37 degrees C before adding to P4CCR5 indicator cell line (HIV-1), ELVIS TM indicator cell line (HSV-1) or HEp2 cell line (RSV) in 96-well microtitre plates. Controls contain virus alone without ET4. The anti-HIV and anti-HSV activities were quantified by estimating beta-galactosidase expression of the respective indicator cell lines while the anti-RSV activity was determined via an immunofluorescent technique, employing monoclonal mouse antibody against the P-protein of RSV. Toxicity of ET4 to cell lines was evaluated in parallel using either the BrdU incorporation method or the MTT method. The effect of ET4 on the enzymatic activity of HIV-1 reverse transcriptase was also evaluated using a chemiluminescent reverse transcriptase assay. Bioassay-guided fractionation of the whole methanol extract of R. farinacea involved sequential screening of HPLC fractions using a vector-based assay technique. RESULTS: ET4 inhibited HSV-1 and RSV potently (IC(50)=6.09 and 3.65 microg/ml, respectively). Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle and the entry step of the RSV replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 reverse transcriptase with an IC(50) of 0.022 microg/ml. Bioassay-guided fractionation of ET4 led to the identification sub-fraction rfO, with activity against lentiviral vector and HIV-1 (RNA viruses) but not against HSV-1 (DNA virus) and sub-fraction rfM, with activity against HSV-1 but not against the lentiviral vector. CONCLUSIONS: ET4 represents a novel fraction from the lichen R. farinacea with broad spectrum antiviral activity against DNA viruses (adenovirus and HSV-1) and RNA viruses (HIV-1 and RSV). The effect against DNA and RNA viruses is mediated by different sub-fractions within R. farinacea.
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Antivirales/farmacología , Líquenes/química , VIH-1/efectos de los fármacos , VIH-1/fisiología , Herpesvirus Humano 1/efectos de los fármacos , Virus Sincitiales Respiratorios/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Gross primary production (GPP) is the primary source of all carbon fluxes in the ecosystem. Understanding variation in this flux is vital to understanding variation in the carbon sink of forest ecosystems, and this would serve as input to forest production models. Using GPP derived from eddy-covariance (EC) measurements, it is now possible to determine the most important factor to scale GPP across sites. We use long-term EC measurements for six coniferous forest stands in Europe, for a total of 25 site-years, located on a gradient between southern France and northern Finland. Eddy-derived GPP varied threefold across the six sites, peak ecosystem leaf area index (LAI) (all-sided) varied from 4 to 22 m(2) m(-2) and mean annual temperature varied from -1 to 13 degrees C. A process-based model operating at a half-hourly time-step was parameterized with available information for each site, and explained 71-96% in variation between daily totals of GPP within site-years and 62% of annual total GPP across site-years. Using the parameterized model, we performed two simulation experiments: weather datasets were interchanged between sites, so that the model was used to predict GPP at some site using data from either a different year or a different site. The resulting bias in GPP prediction was related to several aggregated weather variables and was found to be closely related to the change in the effective temperature sum or mean annual temperature. High R(2)s resulted even when using weather datasets from unrelated sites, providing a cautionary note on the interpretation of R(2) in model comparisons. A second experiment interchanged stand-structure information between sites, and the resulting bias was strongly related to the difference in LAI, or the difference in integrated absorbed light. Across the six sites, variation in mean annual temperature had more effect on simulated GPP than the variation in LAI, but both were important determinants of GPP. A sensitivity analysis of leaf physiology parameters showed that the quantum yield was the most influential parameter on annual GPP, followed by a parameter controlling the seasonality of photosynthesis and photosynthetic capacity. Overall, the results are promising for the development of a parsimonious model of GPP.
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Clima , Geografía , Modelos Biológicos , Tracheophyta/crecimiento & desarrollo , Carbono/metabolismo , Ecosistema , Europa (Continente) , Fotosíntesis , Hojas de la Planta/anatomía & histología , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/fisiología , Temperatura , Tracheophyta/anatomía & histología , Tracheophyta/fisiología , Árboles/anatomía & histología , Árboles/crecimiento & desarrollo , Árboles/fisiologíaRESUMEN
Peatlands are poorly represented in global Earth system modeling frameworks. Here we add a peatland-specific land surface hydrology module (PEAT-CLSM) to the Catchment Land Surface Model (CLSM) of the NASA Goddard Earth Observing System (GEOS) framework. The amended TOPMODEL approach of the original CLSM that uses topography characteristics to model catchment processes is discarded, and a peatland-specific model concept is realized in its place. To facilitate its utilization in operational GEOS efforts, PEAT-CLSM uses the basic structure of CLSM and the same global input data. Parameters used in PEAT-CLSM are based on literature data. A suite of CLSM and PEAT-CLSM simulations for peatland areas between 40°N and 75°N is presented and evaluated against a newly compiled data set of groundwater table depth and eddy covariance observations of latent and sensible heat fluxes in natural and seminatural peatlands. CLSM's simulated groundwater tables are too deep and variable, whereas PEAT-CLSM simulates a mean groundwater table depth of -0.20 m (snow-free unfrozen period) with moderate temporal fluctuations (standard deviation of 0.10 m), in significantly better agreement with in situ observations. Relative to an operational CLSM version that simply includes peat as a soil class, the temporal correlation coefficient is increased on average by 0.16 and reaches 0.64 for bogs and 0.66 for fens when driven with global atmospheric forcing data. In PEAT-CLSM, runoff is increased on average by 38% and evapotranspiration is reduced by 19%. The evapotranspiration reduction constitutes a significant improvement relative to eddy covariance measurements.
RESUMEN
In humans, distinct processes within the hippocampus and rhinal cortex support declarative memory formation. But do these medial temporal lobe (MTL) substructures directly cooperate in encoding new memories? Phase synchronization of gamma-band electroencephalogram (EEG) oscillations (around 40 Hz) is a general mechanism of transiently connecting neural assemblies. We recorded depth-EEG from within the MTL of epilepsy patients performing a memorization task. Successful as opposed to unsuccessful memory formation was accompanied by an initial elevation of rhinal-hippocampal gamma synchronization followed by a later desynchronization, suggesting that effective declarative memory formation is accompanied by a direct and temporarily limited cooperation between both MTL substructures.
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Relojes Biológicos/fisiología , Sincronización Cortical , Corteza Entorrinal/fisiología , Hipocampo/fisiología , Memoria/fisiología , Vías Nerviosas/fisiología , Neuronas/fisiología , Potenciales de Acción/fisiología , Adulto , Corteza Entorrinal/anatomía & histología , Epilepsia del Lóbulo Temporal/fisiopatología , Epilepsia del Lóbulo Temporal/psicología , Femenino , Hipocampo/anatomía & histología , Humanos , Angiografía por Resonancia Magnética , Masculino , Trastornos de la Memoria/etiología , Trastornos de la Memoria/fisiopatología , Trastornos de la Memoria/psicología , Vías Nerviosas/anatomía & histología , Pruebas Neuropsicológicas , Transmisión Sináptica/fisiología , Conducta Verbal/fisiologíaRESUMEN
Although the global prevalence of respiratory syncytial virus (RSV) infection, especially among infants and young children is on the increase, there are only limited therapeutic options for treatment of this disease. Therefore, the search for novel antiviral inhibitors of RSV has become more intensive. In a pilot screening of eighteen compounds from various Aglaia species for anti-RSV activity, we identified dammarenolic acid (ignT1), aglaiol (dupT1) and niloticin (cucT1) as potential anti-RSV compounds, with ignT1 being the most potent. Methylation of ignT1 results in a complete loss of anti-RSV activity. Time of addition studies reveal that both ignT1 and dupT1 target the RSV replication at a post-entry stage, although ignT1 was more potent. Dammarenolic acid (ignT1) was also more cytotoxic than aglaiol (dupT1). By carrying out parallel anti-RSV screening with aphidicolin (a highly cytotoxic diterpenoid) and ignT1, we showed that although aphidicolin was more cytotoxic than ignT1, it had virtually no anti-RSV activity. Therefore, dammarenolic acid, aglaiol and niloticin demonstrate potent anti-RSV activity that shouldbe explored further in the current search for anti-RSV therapeutic agents.
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Aglaia/química , Antivirales/farmacología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacología , Antivirales/aislamiento & purificación , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunohistoquímica , Inflamación/patología , Cinética , Sales de Tetrazolio , Tiazoles , Ensayo de Placa ViralRESUMEN
Concentrations of atmospheric carbon dioxide (CO2) have continued to increase whereas atmospheric deposition of sulphur and nitrogen has declined in Europe and the USA during recent decades. Using time series of flux observations from 23 forests distributed throughout Europe and the USA, and generalised mixed models, we found that forest-level net ecosystem production and gross primary production have increased by 1% annually from 1995 to 2011. Statistical models indicated that increasing atmospheric CO2 was the most important factor driving the increasing strength of carbon sinks in these forests. We also found that the reduction of sulphur deposition in Europe and the USA lead to higher recovery in ecosystem respiration than in gross primary production, thus limiting the increase of carbon sequestration. By contrast, trends in climate and nitrogen deposition did not significantly contribute to changing carbon fluxes during the studied period. Our findings support the hypothesis of a general CO2-fertilization effect on vegetation growth and suggest that, so far unknown, sulphur deposition plays a significant role in the carbon balance of forests in industrialized regions. Our results show the need to include the effects of changing atmospheric composition, beyond CO2, to assess future dynamics of carbon-climate feedbacks not currently considered in earth system/climate modelling.
RESUMEN
We demonstrated a novel application of transient coulostatic pulse technique for the detection of label free DNA hybridization on nm-sized gold interdigitated ultramicroelectrode arrays (Au-IDA) made in silicon technology. The array consists of eight different positions with an Au-IDA pair at each position arranged on the Si-based Biochip. Immobilization of capture probes onto the Au-IDA was accomplished by self-assembling of thiol-modified oligonucleotides. Target hybridization was indicated by a change in the magnitude of the time dependant potential relaxation curve in presence of electroactive Fe(CN)(6)(3-) in the phosphate buffer solution. While complementary DNA hybridization showed 50% increase in the relaxation potential, the non-complementary DNA showed a negligible change. A constant behaviour was noted for all positions. The dsDNA specific intercalating molecule, methylene blue, was found to be enhancing the discrimination effect. The changes in the relaxation potential curves were further corroborated following the ELISA like experiments using ExtraAvidine alkaline phosphatase labelling and redox recycling of para-aminophenol phosphate at IDAs. The coulostatic pulse technique was shown to be useful for identifying DNA sequences from brain tumour gene CK20, human herpes simplex virus, cytomegalovirus, Epstein-Barr virus and M13 phage. Compared to the hybridization of short chain ONTs (27 mers), the hybridization of long chain M13 phage DNA showed three times higher increase in the relaxation curves. The method is fast enough to monitor hybridization interactions in milli or microsecond time scales and is well suitable for miniaturization and integration compared to the common impedance techniques for developing capacitative DNA sensors.
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ADN/análisis , ADN/genética , Electroquímica/instrumentación , Hibridación in Situ/instrumentación , Microelectrodos , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Electroquímica/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Hibridación in Situ/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Electricidad EstáticaRESUMEN
In turkey erythrocytes, the hormone-induced, diuretic-sensitive components of both unidirectional K+ influx and net salt uptake were Cl- dependent, with Br- partially able, and NO3(-) and SO42(-) unable to substitute as the main anion. Since the measured fluxes involve involve Na+/+ cotransport, these observations indicate that the unifying concept of an Na+/K+/2Cl- cotransport system (Geck, P., Pietrzyk, C., Burckhardt, B.-C., Pfeiffer, B. and Heinz, E. (1980) Biochim. Biophys. Acta 600, 432-447) may be extended to hormone-induced ion transport in avian erythrocytes.
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Eritrocitos/metabolismo , Hormonas/sangre , Potasio/sangre , Pavos/sangre , Animales , Bromo/sangre , Cloruros/sangre , Femenino , Nitritos/sangre , Rubidio/sangre , Sodio/sangre , Sulfitos/sangreRESUMEN
(1) The occurrence of volume-regulatory responses in isolated rat hepatocytes was investigated. After hypotonic shock, cells were able to shrink back to their normal volume within 15 min. By contrast, after hypertonic shock cells kept their shrunken volume for at least 15 min. (2) Na+-alanine cotransport increases K+ permeability in rat hepatocytes (Kristensen, L.O. (1980) J. Biol. Chem. 255, 5236-5243). It is shown here that this effect is due to cell swelling secondary to alanine accumulation rather than to the Na+-alanine cotransport itself.
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Líquidos Corporales/fisiología , Líquido Intracelular/fisiología , Hígado/citología , Potasio/metabolismo , Alanina/metabolismo , Animales , Transporte Biológico , Técnicas In Vitro , Hígado/metabolismo , Masculino , Permeabilidad , Ratas , Ratas EndogámicasRESUMEN
Potassium self-exchange in ascites cells was partially suppressed when chloride in the incubation medium was replaced by bromide, and completely abolished in iodide-, nitrate- or sulfate-containing saline. Thus, it appears that potassium self-exchange activity is anion-dependent.
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Aniones/farmacología , Potasio/metabolismo , Animales , Ascitis/metabolismo , Transporte Biológico/efectos de los fármacos , Cloruros/farmacología , Furosemida/farmacología , Ratones , Nitratos/farmacología , Ouabaína/farmacología , Rubidio/metabolismoRESUMEN
As turkey erythrocytes were progressively depleted of ATP by preincubation with dinitrophenol, the (Na+ + K+ + 2Cl-)-cotransport system (assayed by the bumetanide-sensitive fraction of 86Rb+ influx) became less responsive to activation. The dependence upon intracellular ATP concentration was significantly steeper for transport activated by hypertonic shock (halfmaximal activity at 0.7 mM ATP) than for that activated by either epinephrine or cyclic AMP (halfmaximal activity at 1.7 mM ATP). Upon removal of epinephrine or cyclic AMP from cells that had been preincubated with those substances, bumetanide-sensitive transport activity declined sharply, even though the intracellular cyclic AMP concentration was still over 10-fold that required to maximally activate the transport system. These data are in agreement with the notion that the (Na+ + K+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by cyclic AMP, presumably through the 'classical' pathway involving a protein kinase. They do however indicate that some other, as yet undefined aspect of cyclic AMP metabolism is important for the maintenance of transport activity.
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Adenosina Trifosfato/fisiología , Cloruros/sangre , AMP Cíclico/farmacología , Eritrocitos/metabolismo , Potasio/sangre , Sodio/sangre , Animales , Transporte Biológico/efectos de los fármacos , Bumetanida/farmacología , AMP Cíclico/sangre , Epinefrina/farmacología , Femenino , Técnicas In Vitro , Presión Osmótica , PavosRESUMEN
The bumetanide-sensitive (K+ + Na+ + 2Cl-)-cotransport system in turkey erythrocytes is activated by either of two treatments: addition of epinephrine or an increase in osmolarity. At elevated (20 mM) K+ concentration, cotransport activity induced by epinephrine slowly (within 90 min) declines to background level again. This time-dependent inactivation has been linked to bumetanide-sensitive cell swelling. We have compared both the initial rate of cotransport activity and its time dependence after induction by either epinephrine, increased osmolarity or a combination of the two treatments. As a measure of cotransport activity we took the bumetanide-sensitive fraction of 86Rb+ influx. Immediately after activation, several kinetic characteristics of this flux (Vmax; Km towards K+; Ki towards bumetanide; pH profile) were identical in cells activated by either treatment. By contrast, cotransport activated by hypertonicity was significantly more resistant towards subsequent inactivation. We show this to be due to the increase in intracellular ion concentrations brought about by hypertonic cell shrinkage. This tended to reverse the driving force for cotransport, and thereby prevented the bumetanide-sensitive swelling associated with inactivation. Our data support the notion that cell volume plays a key role both in the activation and in the time-dependent inactivation of bumetanide-sensitive transport.
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Bumetanida/farmacología , Diuréticos/farmacología , Eritrocitos/citología , Potasio/sangre , Animales , Transporte Biológico Activo/efectos de los fármacos , Epinefrina/farmacología , Membrana Eritrocítica/metabolismo , Cinética , Matemática , Concentración Osmolar , PavosRESUMEN
Steady-state fluxes of 86Rb+ (as a tracer for K+) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247--1249). Analogous to previous findings in ascites tumor cells, 86Rb+ influx in the parent strain was differentiated into a ouabain-inhibitable 'pump' flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual 'leak' flux. On the basis of this flux characterization, 86Rb+ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, 86Rb+ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K+ in the steady state is independent of the ordering of membrane lipid acyl chains.
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Colesterol/fisiología , Lípidos de la Membrana/fisiología , Rubidio/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Cricetinae , Femenino , Furosemida/farmacología , Cinética , Metilglucósidos/metabolismo , Ouabaína/farmacología , Ovario , Potasio/metabolismoRESUMEN
Cells of Entamoeba histolytica accumulated K+ and extruded Na+ compared to the concentrations of those ions present in the growth medium. Pinocytic activity, measured by the uptake of horseradish peroxidase of 125I-polyvinylpyrrolidone, was high (up to 0.3 ml/ml cells per h). Upon addition of cytochalasin B, at a concentration (20 microM) that completely blocked pinocytosis, cells lost up to 40% of their Na+ content within 90 min; K+ content was not affected or increased slightly compared to control cells without the inhibitor. Cation loss was associated with cell shrinkage. The dose-response curves for the effects of cytochalasin B on pinocytosis and Na+ content were identical. These data provide direct evidence that pinocytosis is an important component of the homeostatic system for Na+.
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Citocalasina B/farmacología , Entamoeba histolytica/efectos de los fármacos , Sodio/metabolismo , Equilibrio Hidroelectrolítico/efectos de los fármacos , Animales , Endocitosis/efectos de los fármacos , Entamoeba histolytica/citología , Pinocitosis/efectos de los fármacosRESUMEN
Unidirectional potassium influx in simian virus-transformed 3T3 cells was dissected into a ouabain-inhibitable "pump' component, a bumetanide-sensitive and chloride-dependent "cotransport' component, and a residual "leak' flux. The bumetanide-sensitive component was stimulated 2-3-fold by a 60-min preincubation with ouabain. Subsequent washing of the cells and incubation in ouabain-free saline reversed both the inhibition of the Na+ pump and the stimulation of bumetanide-sensitive flux. Bumetanide-sensitive potassium influx was also stimulated by hypertonic cell shrinkage (induced by 0.1 M or 0.2 M sorbitol). This latter observation suggests that the bumetanide-sensitive system may play a role in cellular volume regulation.