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In this study, no difference in bone loss was observed between patients with early RA initially treated with COmbinatietherapie Bij Reumatoide Artritis (COBRA) (including initially 60 mg/day prednisolone) and patients treated with COBRA-light (including initially 30 mg/day prednisolone) during 4-year observation. PURPOSE: To assess changes in bone mineral density (BMD) after 4 years in early rheumatoid arthritis (RA) patients initially treated with COBRA-light or COBRA therapy. METHODS: In a 1 year, open-label, randomised, non-inferiority trial, patients were assigned to COBRA-light (methotrexate 25 mg/week plus initially prednisolone 30 mg/day) or COBRA (methotrexate 7.5 mg/week, sulfasalazine 2 g/day plus initially prednisolone 60 mg/day) therapy. After 1 year, antirheumatic treatment was at the discretion of treating rheumatologists. BMD was measured at baseline and after 1, 2 and 4 years at hips and lumbar spine with dual-energy X-ray absorptiometry. BMD changes between treatment strategies on average over time were compared with GEE analysis. RESULTS: Data from 155 out of 162 patients could be analysed: 68% were female with a mean age of 52 (SD 13) years. Both COBRA-light and COBRA therapy showed declines in BMD at the total hip of -3.3% and -1.7%, respectively (p = 0.12), and the femoral neck, -3.7% and -3.0%, respectively (p = 0.95). At the lumbar spine, both treatment groups showed minor decline in BMD over 4 years: -0.5% and -1.0%, respectively (p = 0.10). CONCLUSION: In a treat-to-target design in early RA, over 4 years, no differences between groups were found in change in BMD at total hip, femoral neck and the lumbar spine. At the hip, bone loss was around 3% in both groups, while mild bone loss was observed at lumbar spine, both in patients starting prednisolone 60 and 30 mg/day. These data suggest that the well-known negative effects of prednisolone can be modulated by modern treatment of RA.
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Antirreumáticos , Artritis Reumatoide , Absorciometría de Fotón , Antirreumáticos/efectos adversos , Artritis Reumatoide/tratamiento farmacológico , Densidad Ósea , Quimioterapia Combinada , Femenino , Humanos , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Prednisolona/efectos adversosRESUMEN
Elevated environmental carbon dioxide (pCO2) levels have been found to cause organ damage in the early life stages of different commercial fish species, including Atlantic cod (Gadus morhua). To illuminate the underlying mechanisms causing pathologies in the intestines, the kidney, the pancreas and the liver in response to elevated pCO2, we examined related gene expression patterns in Atlantic cod reared for two months under three different pCO2 regimes: 380 µatm (control), 1800 µatm (medium) and 4200 µatm (high). We extracted RNA from whole fish sampled during the larval (32 dph) and early juvenile stage (46 dph) for relative expression analysis of 18 different genes related to essential metabolic pathways. At 32 dph, larvae subjected to the medium treatment displayed an up-regulation of genes mainly associated with fatty acid and glycogen synthesis (GYS2, 6PGL, ACoA, CPTA1, FAS and PPAR1b). Larvae exposed to the high pCO2 treatment upregulated fewer but similar genes (6PGL, ACoA and PPAR1b,). These data suggest stress-induced alterations in the lipid and fatty acid metabolism and a disrupted lipid homeostasis in larvae, providing a mechanistic link to the findings of lipid droplet overload in the liver and organ pathologies. At 46 dph, no significant differences in gene expression were detected, confirming a higher resilience of juveniles in comparison to larvae when exposed to elevated pCO2 up to 4200 µatm.
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Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Gadus morhua/genética , Gadus morhua/metabolismo , Animales , Cambio Climático , Concentración de Iones de Hidrógeno , Larva , Metabolismo de los Lípidos , Océanos y Mares , Agua de MarRESUMEN
Research on the thermal biology of Antarctic marine organisms has increased awareness of their vulnerability to climate change, as a flipside of their adaptation to life in the permanent cold and their limited capacity to acclimate to variable temperatures. Here, we employed a species-specific microarray of the Antarctic eelpout, Pachycara brachycephalum, to identify long-term shifts in gene expression after 2 months of acclimation to six temperatures between -1 and 9 °C. Changes in cellular processes comprised signalling, post-translational modification, cytoskeleton remodelling, metabolic shifts and alterations in the transcription as well as translation machinery. The magnitude of transcriptomic responses paralleled the change in whole animal performance. Optimal growth at 3 °C occurred at a minimum in gene expression changes indicative of a balanced steady state. The up-regulation of ribosomal transcripts at 5 °C and above was accompanied by the transcriptomic activation of differential protein degradation pathways, from proteasome-based degradation in the cold towards lysosomal protein degradation in the warmth. From 7 °C upwards, increasing transcript levels representing heat-shock proteins and an acute inflammatory response indicate cellular stress. Such patterns may contribute to a warm-induced energy deficit and a strong weight loss at temperatures above 6 °C. Together, cold or warm acclimation led to specific cellular rearrangements and the progressive development of functional imbalances beyond the optimum temperature. The observed temperature-specific expression profiles reveal the molecular basis of thermal plasticity and refine present understanding of the shape and positioning of the thermal performance curve of ectotherms on the temperature scale.
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Aclimatación/genética , Perciformes/genética , Temperatura , Transcriptoma , Animales , Regiones Antárticas , Femenino , Proteínas de Choque Térmico/metabolismo , Inflamación/metabolismo , Hígado/metabolismo , Masculino , Estrés Oxidativo , Perciformes/crecimiento & desarrollo , Biosíntesis de Proteínas , Proteolisis , Transducción de Señal , Regulación hacia ArribaRESUMEN
BACKGROUND: A serious game called SPARX (Smart, Positive, Active, Realistic, X-factor thoughts), originally developed in New Zealand and incorporating cognitive behavioural therapy (CBT) principles, has been shown to help reduce symptoms of depression and anxiety in adolescents with mild to moderate depression in studies undertaken in Australasia. However, SPARX has never been trialled in the United Kingdom (UK), and there have been issues relating to low engagement when it has been used in a real-world context. AIMS: To conduct the first pilot and feasibility randomised controlled trial (RCT) in England to explore the use of SPARX in different settings. The trial will explore whether SPARX supported by an e-coach (assistant psychologists) improves adherence and engagement compared with self-directed (i.e. self-help) use. The trial results will be used to inform the optimal mode of delivery (SPARX supported vs. SPARX self-directed), to calculate an appropriate sample size for a full RCT, and to decide which setting is most suitable. METHODS: Following consultation with young people to ensure study suitability/appropriateness, a total of 120 adolescents (11-19 years) will be recruited for this three-arm study. Adolescents recruited for the study across England will be randomised to receive either SPARX with human support (from an e-coach), self-directed SPARX, or a waitlist control group. Assessments will be conducted online at baseline, week 4, and 8-10-week post-randomisation. The assessments will include measures which capture demographic, depression (Patient Health Questionnaire modified for adolescents [PHQ-A]) and anxiety (Revised Child Anxiety and Depression Scale [RCADS]) symptomatology, and health-related quality-of-life data (EQ-5D-Y and proxy version). Analyses will be primarily descriptive. Qualitative interviews will be undertaken with a proportion of the participants and clinical staff as part of a process evaluation, and the qualitative data gathered will be thematically analysed. Finally, feasibility data will be collected on recruitment details, overall study uptake and engagement with SPARX, participant retention, and youth-reported acceptability of the intervention. DISCUSSION: The findings will inform the design of a future definitive RCT of SPARX in the UK. If the subsequent definitive RCT demonstrates that SPARX is effective, then an online serious game utilising CBT principles ultimately has the potential to improve the provision of care within the UK's health services if delivered en masse. TRIAL REGISTRATION: ISRCTN: ISRCTN15124804. Registered on 16 January 2023, https://www.isrctn.com/ISRCTN15124804 .
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Polar ecosystems are experiencing amongst the most rapid rates of regional warming on Earth. Here, we discuss 'omics' approaches to investigate polar biodiversity, including the current state of the art, future perspectives and recommendations. We propose a community road map to generate and more fully exploit multi-omics data from polar organisms. These data are needed for the comprehensive evaluation of polar biodiversity and to reveal how life evolved and adapted to permanently cold environments with extreme seasonality. We argue that concerted action is required to mitigate the impact of warming on polar ecosystems via conservation efforts, to sustainably manage these unique habitats and their ecosystem services, and for the sustainable bioprospecting of novel genes and compounds for societal gain.
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Ecosistema , Multiómica , Biodiversidad , PredicciónRESUMEN
Heat-shock proteins have been shown to be critical antigens in a number of autoimmune diseases. In human arthritis and in experimentally induced arthritis in animals, disease development was seen to coincide with development of immune reactivity directed against not only bacterial hsp60, but also against its mammalian homologue. We have developed murine monoclonal antibodies after immunization with recombinant human hsp60. Antibodies with unique specificity for mammalian hsp60, not crossreactive with the bacterial counterpart (LK1), and antibodies recognizing both human and bacterial hsp60 (LK2) were selected. Both antibodies recognize epitopes located between amino acid positions 383 and 447 of human hsp60. In immunogold electron microscopy, the mitochondrial localization of hsp60 in HepG2 cells was shown. Furthermore, both LK1 and LK2 showed a raised level of staining in light microscopy immunohistochemistry of synovial membranes in patients with juvenile chronic arthritis. The increased staining for LK1, with a unique specificity for mammalian hsp60, thus unequivocally demonstrates that this is due to a raised level of expression of endogenously produced host hsp60 and not to deposition of bacterial antigens.
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Anticuerpos Monoclonales , Artritis Juvenil/patología , Proteínas de Choque Térmico/análisis , Membrana Sinovial/patología , Western Blotting , Carcinoma Hepatocelular , Línea Celular , Niño , Deleción Cromosómica , Electroforesis en Gel de Poliacrilamida , Proteínas de Choque Térmico/genética , Humanos , Inmunohistoquímica , Neoplasias Hepáticas , Microscopía Inmunoelectrónica , Peso Molecular , Proteínas Recombinantes , Valores de Referencia , TransfecciónRESUMEN
Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).
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Bordetella pertussis/química , Escherichia coli/química , Proteínas Quinasas/química , Señales de Clasificación de Proteína/química , Transducción de Señal , Cromatografía en Gel , Dimerización , Histidina Quinasa , Peso Molecular , Fosforilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie , UltracentrifugaciónRESUMEN
Ocean warming and acidification are threatening marine ecosystems. In marine animals, acidification is thought to enhance ion regulatory costs and thereby baseline energy demand, while elevated temperature also increases baseline metabolic rate. Here we investigated standard metabolic rates (SMR) and plasma parameters of Atlantic cod (Gadus morhua) after 3-4 weeks of exposure to ambient and future PCO2 levels (550, 1200 and 2200 µatm) and at two temperatures (10, 18 °C). In vivo branchial ion regulatory costs were studied in isolated, perfused gill preparations. Animals reared at 18 °C responded to increasing CO2 by elevating SMR, in contrast to specimens at 10 °C. Isolated gills at 10 °C and elevated PCO2 (≥1200 µatm) displayed increased soft tissue mass, in parallel to increased gill oxygen demand, indicating an increased fraction of gill in whole animal energy budget. Altered gill size was not found at 18 °C, where a shift in the use of ion regulation mechanisms occurred towards enhanced Na(+)/H(+)-exchange and HCO3 (-) transport at high PCO2 (2200 µatm), paralleled by higher Na(+)/K(+)-ATPase activities. This shift did not affect total gill energy consumption leaving whole animal energy budget unaffected. Higher Na(+)/K(+)-ATPase activities in the warmth might have compensated for enhanced branchial permeability and led to reduced plasma Na(+) and/or Cl(-) concentrations and slightly lowered osmolalities seen at 18 °C and 550 or 2200 µatm PCO2 in vivo. Overall, the gill as a key ion regulation organ seems to be highly effective in supporting the resilience of cod to effects of ocean warming and acidification.
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Aclimatación , Ecosistema , Metabolismo Energético , Gadus morhua/metabolismo , Branquias/metabolismo , Transporte Iónico , Temperatura , Animales , Bicarbonatos/sangre , Biomarcadores/sangre , Cloruros/sangre , Femenino , Gadus morhua/sangre , Concentración de Iones de Hidrógeno , Masculino , Océanos y Mares , Concentración Osmolar , Consumo de Oxígeno , Sodio/sangre , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The hydrolysis of cocaine and its N-demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.
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Cocaína/metabolismo , Hígado/enzimología , Hidrolasas de Éster Carboxílico , Colinesterasas/sangre , Cocaína/análogos & derivados , Remoción de Radical Alquila , Humanos , Hidrólisis , Técnicas In Vitro , CinéticaRESUMEN
Color constancy was studied by the method of comparing color samples under two different illuminants using a CRT color monitor. In addition to the classical approach in which one of the illuminants is a (standard) white, we performed experiments in which the range of differential illumination was extended by using pairs of lights that were both colored. The stimulus pattern consisted of an array of thirty-five color samples (including five neutral samples) on a white background. A trichromatic illuminant-object interaction was simulated analogous to that resulting from illumination by three monochromatic lights. The test samples, as seen under "test" and "match" illumination, were successively presented to the left and right eye (haploscopic matching). The data show systematic deviations from predictions on the basis of cone-specific normalization procedures like those incorporated in the Retinex algorithm and the von Kries transformation. The results can be described by a nonlinear response transformation that depends on two factors, receptor-specific sample/background contrast and the extent to which the illuminant stimulates the receptor system in question. The latter factor explains the deviations. These are mainly caused by the short-wave-sensitive system, as a consequence of the fact that this system can be more selectively stimulated than the other, spectrally less separated, cone systems.
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Percepción de Color/fisiología , Células Fotorreceptoras/fisiología , Humanos , Luz , Matemática , Modelos PsicológicosRESUMEN
The present study demonstrates that an object embedded in an array of identical objects can pop-out. Dependent on the stimuli preceding the search display, local (chromatic) adaptation causes an identical object to pop-out because it appears to have a colour (Expt 1) or brightness (Expt 2) that is slightly different from the colour and brightness of the other objects in the display. Experiment 3 shows that this pop-out even occurs when the stimulus preceding the search display is presented for only 100 msec.
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Adaptación Ocular/fisiología , Percepción de Color/fisiología , Postimagen/fisiología , Humanos , Masculino , Tiempo de Reacción , Factores de TiempoRESUMEN
Color constancy was studied under conditions simulating either natural or extremely artificial illumination. Four test illuminants were used: two broadband phases of daylight (correlated color temperatures 4000 and 25,000 K) and two spectrally impoverished metamers of these lights, each consisting of only two wavelengths. A computer controlled color monitor was used for reproducing the chromaticities and luminances of an array of Munsell color samples rendered under these illuminants. An asymmetric haploscopic matching paradigm was used in which the same stimulus pattern, either illuminated by one of the test illuminants, or by a standard broadband daylight (D65), was alternately presented to the left and right eye. Subjects adjusted the RGB settings of the samples seen under D65 (match condition), to match the appearance of the color samples seen under the test illuminant. The results show the expected failure of color constancy under two-wavelengths illumination, and approximate color constancy under natural illumination. Quantitative predictions of the results were made on the basis of two different models, a computational model for recovering surface reflectance, and a model that assumes the color response to be determined by cone-specific contrast and absolute level of stimulation (Lucassen & Walraven, 1993). The latter model was found to provide somewhat more accurate predictions, under all illuminant conditions.
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Percepción de Color/fisiología , Iluminación , Humanos , Masculino , Matemática , Modelos Biológicos , Espectrofotometría , Visión MonocularAsunto(s)
Artritis Experimental/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Células Clonales , Proteínas de Choque Térmico/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Linfocitos , Mycobacterium tuberculosis/inmunología , Péptidos/inmunología , Ratas , Ratas Endogámicas Lew , Receptores de Antígenos de Linfocitos T alfa-beta/química , Linfocitos T/patologíaAsunto(s)
Antipirina/metabolismo , Hígado/enzimología , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas/metabolismo , Animales , Antipirina/orina , Estabilidad de Medicamentos , Masculino , Metilcolantreno/farmacología , Fenobarbital/farmacología , Ratas , Factores Sexuales , Factores de TiempoRESUMEN
Exposure of ectothermic organisms to variations in temperatures causes a transient mismatch between energy supply and demand, which needs to be compensated for during acclimation. Adenosine accumulation from ATP breakdown indicates such an imbalance and its reversal reflects a restoration of energy status. We monitored adenosine levels in blood serum and liver of common eelpout (Zoarces viviparus) during cold exposure in vivo. Furthermore, we tested its effect on the pattern of thermal acclimation in hepatocytes isolated from cold- (4 degrees C) versus warm- (11 degrees C) exposed fish. Adenosine levels increased during cold exposure in vivo and reached a transient maximum after 24 h in serum, but remained permanently elevated in liver. Whole animal cold acclimation induced a rise of liver citrate synthase activity by 44+/-15%, but left cytochrome c oxidase activity (COX) and RNA expression of the respective genes unchanged. Cold incubation of hepatocytes from warm-acclimated fish failed to cause an increase of mitochondrial enzyme activities despite increased COX4 mRNA levels. Conversely, warm acclimation of hepatocytes from cold-acclimated fish reduced both enzyme activities and COX2 and COX4 mRNA levels by 26-37%. Adenosine treatment of both warm- and cold-acclimated hepatocytes suppressed COX activities but activated COX mRNA expression. These effects were not receptor mediated. The present findings indicate that adenosine has the potential to regulate mitochondrial functioning in vivo, albeit the pathways resulting in the contrasting effects on expression and activity need to be identified.
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Adenosina/sangre , Frío , Complejo IV de Transporte de Electrones/metabolismo , Perciformes/sangre , Aclimatación/efectos de los fármacos , Adenosina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Complejo IV de Transporte de Electrones/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hígado/enzimología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.
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Adjustments in mitochondrial properties and capacities are crucial in acclimatization to seasonal cold as well as in evolutionary cold adaptation of marine ectotherms. To examine whether gene expression mechanisms contribute to different settings of aerobic capacities in populations of cod (Gadus morhua) along a latitudinal cline, maximum activities of key enzymes of mitochondrial metabolism and their respective mRNA levels were compared in white muscle and liver of cold (4 degrees C) and warm (10 degrees C) acclimated individuals from cod populations of the North Sea and the Barents Sea, respectively. In white muscle, cold acclimation caused a parallel increase in citrate synthase (CS) and in cytochrome c oxidase (COX) activities, but with a much larger effect in the cold eurythermal Arctic population. In liver, cold acclimation was accompanied by increments in CS activities, but differences between populations were minor. Overall COX activities in liver were not affected by cold acclimation, but were higher in the cold adapted population. In both populations increments in muscle CS capacities were tightly correlated with elevated mRNA levels, suggesting transcriptional control of citrate synthase levels in muscle. In liver, CS mRNA levels differed between populations but were not affected by cold acclimation, so that post-transcriptional control may contribute to elevated functional levels in this tissue. Mitochondrial-encoded COX2 mRNA levels were not limiting for functional activities in both tissues, in favour of post-transcriptional control or limitations by other transcripts of the COX complex. Altogether, the differentiation in gene expression between both populations was more strongly expressed at 4 degrees C. The comparison of functional levels and transcript levels may reflect genetic differentiation at functional sites, in line with genetic differences between the two populations previously established by non-coding genetic markers.
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Aclimatación , Clima , Frío , Gadus morhua/fisiología , Mitocondrias/enzimología , Animales , Regiones Árticas , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Proteínas de Peces/metabolismo , Gadus morhua/genética , Regulación de la Expresión Génica , Hígado/enzimología , Músculos/enzimología , Mar del Norte , Océanos y Mares , ARN Mensajero/metabolismoRESUMEN
Studies were undertaken to investigate the Ni(II)-binding properties of human blood serum and to identify the low-molecular-weight Ni(II)-binding constitutents in the serum. Three Ni(II)-binding fractions were obtained when labeled nickel chloride (63NiCl2) was added to the native serum. Of the total Ni(II), 95.7% was associated with albumin, 4.2% was bound to low-molecular-weight components, and a small fraction, usually less than 0.1% was associated with a high-molecular-weight protein that was eluted in the void volume of Sephadex G-150. Amino acids were shown to be responsible for the low-molecular-weight Ni(II)-binding fraction and L-histidine was found to be the main (Ni(II)-binding amino acid in human blood serum. Compared with albumin, L-histidine was shown to possess a greater affinity for Ni(II). Ni(II)-binding to human albumin became evident only when no more L-histidine was available. Since the concentration of albumin is much higher than the concentration of L-histidine in normal serum, most of the added Ni(II) was associated with albumin. The equilibria between Ni(II)-L-histidine and Ni(II)-albumin may facilitate the transport of Ni(ii) between blood and tissues.
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Proteínas Sanguíneas/metabolismo , Níquel/sangre , Aminoácidos/análisis , Electroforesis de las Proteínas Sanguíneas , Fenómenos Químicos , Química , Humanos , Peso Molecular , Unión Proteica , UltracentrifugaciónRESUMEN
Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 degrees C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97-106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis-Menten function dependent on the catalytic properties of the enzyme (kcat approximately equal to 9200 min-1, Km approximately equal to 0.12 mM), then level off to a slower rate determined by the desorption equilibrium constant (KD approximately equal to 0.01 mM) and desorption rate constant (kD approximately equal to 0.15 min-1). The relaxation time, tau, for the transition to the desorption-limited reaction is approximately 0.5 min; this large value of tau probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lysophosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decrease to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.
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Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Venenos de Crotálidos , Geles , Hidrólisis , Cinética , Matemática , Modelos Biológicos , Fosfatidilcolinas , Fosfolipasas A2RESUMEN
The expression of the kdpFABC operon, coding for the K+-translocating Kdp system, is controlled by the two regulatory proteins, KdpD and KdpE, which belong to the group of sensor kinase/response regulator systems. This study describes the construction and analysis of KdpD sensor kinases, in which different deletions in the N-terminal part of the protein were introduced. Truncated KdpD proteins, in which the membrane-spanning segments were deleted, had lost their phosphorylation capacity. Truncated KdpD proteins, in which the four membrane-spanning helices were untouched, were still phosphorylated, and the phosphoryl group could be transferred to the response regulator KdpE in vitro. Furthermore, these truncated KdpD proteins cause dephosphorylation of KdpE(P), which is comparable with that of the wild-type protein. To investigate the effect of the deletions on signal transduction in vivo the corresponding kdp genes were transferred to the chromosome. Growth studies with the mutant strains are in accord with the data obtained from the in vitro studies. Furthermore, kdp expression was investigated using a KdpA-LacZ fusion. The data obtained support the notion that the extent of kdp expression is modulated by the N-terminal part of KdpD.