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OBJECTIVE: The reconstruction of an input based on a sparse combination of signals, known as sparse coding, has found widespread use in signal processing. In this work, the combination of sparse coding with Kalman filtering is explored and its potential is shown on two use-cases. METHODS: This work extends the Iterative Shrinkage and Thresholding Algorithm with a Kalman filter in the sparse domain. The resulting method may be implemented as a deep unfolded neural network and may be applied to any signal which has a sparse representation and a known or assumed relation between consecutive measurements. This method is evaluated on the use cases of noise reduction in the electrocardiogram (ECG) and the estimation of object motility. RESULTS: For ECG denoising, the proposed method achieved an improvement in Signal-to-Noise ratio of 18.6 dB, which is comparable to state-of-the-art. In motility estimation, a correlation of 0.84 with ground truth simulations was found. CONCLUSION: The proposed method was shown to have advantages over sparse coding and Kalman filtering alone. Due to the low complexity and high generalizability of the proposed method, the implementation of context-specific knowledge or an extension to other applications can be readily made. SIGNIFICANCE: The presented Kalman-ISTA algorithm is a resource-efficient method combining the promise of both sparse coding and Kalman filtering, making it well-suited for various applications.
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Algoritmos , Eletrocardiografia , Processamento de Sinais Assistido por Computador , Razão Sinal-Ruído , Eletrocardiografia/métodos , Humanos , Movimento/fisiologiaRESUMO
OBJECTIVE: Standard surgical treatment of advanced-stage ovarian carcinoma with electrosurgery cannot always result in complete cytoreductive surgery (CRS), especially when many small metastases are found on the mesentery and intestinal surface. We investigated whether adjuvant use of a neutral argon plasma device can help increase the complete cytoreduction rate. PATIENTS AND METHODS: 327 patients with FIGO stage IIIB-IV epithelial ovarian cancer (EOC) who underwent primary or interval CRS were randomized to either surgery with neutral argon plasma (PlasmaJet) (intervention) or without PlasmaJet (control group). The primary outcome was the percentage of complete CRS. The secondary outcomes were duration of surgery, blood loss, number of bowel resections and colostomies, hospitalization, 30-day morbidity, and quality of life (QoL). RESULTS: Complete CRS was achieved in 119 patients (75.8%) in the intervention group and 115 patients (67.6%) in the control group (risk difference (RD) 8.2%, 95% confidence interval (CI) -0.021 to 0.181; P = 0.131). In a per-protocol analysis excluding patients with unresectable disease, complete CRS was obtained in 85.6% in the intervention group and 71.5% in the control group (RD 14.1%, 95% CI 0.042 to 0.235; P = 0.005). Patient-reported QoL at 6 months after surgery differed between groups in favor of PlasmaJet surgery (95% CI 0.455-8.350; P = 0.029). Other secondary outcomes did not differ significantly. CONCLUSIONS: Adjuvant use of PlasmaJet during CRS for advanced-stage ovarian cancer resulted in a significantly higher proportion of complete CRS in patients with resectable disease and higher QoL at 6 months after surgery. (Funded by ZonMw, Trial Register NL62035.078.17.) TRIAL REGISTRATION: Approved by the Medical Ethics Review Board of the Erasmus University Medical Center Rotterdam, the Netherlands, NL62035.078.17 on 20-11-2017. Recruitment started on 30-1-2018.
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Neoplasias Ovarianas , Gases em Plasma , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/cirurgia , Procedimentos Cirúrgicos de Citorredução/métodos , Feminino , Humanos , Países Baixos , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgia , Qualidade de VidaRESUMO
Solid particles adsorbed at fluid interfaces are crucial for the mechanical stability of Pickering emulsions. The key parameter which determines the kinetic and thermodynamic properties of these colloids is the particle contact angle, θ. Several methods have recently been developed to measure the contact angle of individual particles adsorbed at liquid-liquid interfaces, as morphological and chemical heterogeneities at the particle surface can significantly affect θ. However, none of these techniques enables the simultaneous visualization of the nanoparticles and the reconstruction of the fluid interface to which they are adsorbed, in situ. To tackle this challenge, we utilize a newly developed super-resolution microscopy method, called iPAINT, which exploits non-covalent and continuous labelling of interfaces with photo-activatable fluorescent probes. Herewith, we resolve with nanometer accuracy both the position of individual nanoparticles at a water-octanol interface and the location of the interface itself. First, we determine single particle contact angles for both hydrophobic and hydrophilic spherical colloids. These experiments reveal a non-negligible dependence of θ on particle size, from which we infer an effective line tension, τ. Next, we image elliptical particles at a water-decane interface, showing that the corresponding interfacial deformations can be clearly captured by iPAINT microscopy.
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BACKGROUND: The CEAwatch randomized trial showed that follow-up with intensive carcinoembryonic antigen (CEA) monitoring (CEAwatch protocol) was better than care as usual (CAU) for early postoperative detection of colorectal cancer recurrence. The aim of this study was to calculate overall survival (OS) and disease-specific survival (DSS). METHODS: For all patients with recurrence, OS and DSS were compared between patients detected by the CEAwatch protocol versus CAU, and by the method of detection of recurrence, using Cox regression models. RESULTS: Some 238 patients with recurrence were analysed (7·5 per cent); a total of 108 recurrences were detected by CEA blood test, 64 (55·2 per cent) within the CEAwatch protocol and 44 (41·9 per cent) in the CAU group (P = 0·007). Only 16 recurrences (13·8 per cent) were detected by patient self-report in the CEAwatch group, compared with 33 (31·4 per cent) in the CAU group. There was no significant improvement in either OS or DSS with the CEAwatch protocol compared with CAU: hazard ratio 0·73 (95 per cent 0·46 to 1·17) and 0·78 (0·48 to 1·28) respectively. There were no differences in survival when recurrence was detected by CT versus CEA measurement, but both of these methods yielded better survival outcomes than detection by patient self-report. CONCLUSION: There was no direct survival benefit in favour of the intensive programme, but the CEAwatch protocol led to a higher proportion of recurrences being detected by CEA-based blood test and reduced the number detected by patient self-report. This is important because detection of recurrence by blood test was associated with significantly better survival than patient self-report, indirectly supporting use of the CEAwatch protocol.
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Antígeno Carcinoembrionário/metabolismo , Neoplasias do Colo/cirurgia , Proteínas de Neoplasias/metabolismo , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias Retais/cirurgia , Assistência ao Convalescente , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Colo/sangue , Neoplasias do Colo/mortalidade , Detecção Precoce de Câncer/métodos , Métodos Epidemiológicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/mortalidade , Neoplasias Retais/sangue , Neoplasias Retais/mortalidadeRESUMO
Colloidal systems exhibit intriguing assembly phenomena with impact in a wide variety of technological fields. The use of magnetically responsive colloids allows one to exploit interactions with an anisotropic dipolar nature. Here, we reveal magnetic interfacial rotaphoresis - a magnetically-induced rotational excitation that imposes a translational motion on colloids by a strong interaction with a solid-liquid interface - as a means to transport, disperse, and order dense colloidal assemblies. By balancing magnetic dipolar and hydrodynamic interactions at a symmetry-breaking interface, rotaphoresis effectuates a translational dispersive motion of the colloids and surprisingly transforms large and dense multilayer assemblies into single-particle layers with quasi-hexagonal ordering within seconds and with velocities of mm s(-1). We demonstrate the application of interfacial rotaphoresis to enhance molecular target capture, showing an increase of the molecular capture rate by more than an order of magnitude.
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Coloides/química , Magnetismo , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Corantes Fluorescentes/química , Imunoglobulina G/química , Imunoglobulina G/imunologia , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de FluorescênciaRESUMO
The fundamental molecular and microscopic properties of materials leading to dynamic wetting and relaxation effects have been subject to numerous studies in the past decades, but a thorough understanding is still missing. While most previous experiments utilize fluids deposited on planar substrates, this article focuses on an attractive alternative based on single colloidal particles: colloidal particles have the ability to strongly interact with fluid-fluid interfaces and the behavior strongly depends on the surface properties of the particles and the fluids used. Recent progress in the manipulation and synthesis of colloidal particles with well-defined surface properties and shapes makes them ideal candidates to probe the fundamental surface properties leading to dynamic wetting effects. In this paper we review and discuss the status of experimental and numerical techniques to characterize the dynamic wetting of single particles at fluid-fluid interfaces, with the aim to assist scientists and engineers in the design of new experimental techniques and particle-based (bio)analytical tools.
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Molhabilidade , Coloides , Técnicas Analíticas Microfluídicas , Modelos Teóricos , Estudos Prospectivos , Propriedades de SuperfícieRESUMO
Molecular interferences are an important challenge in biotechnologies based on antibody-antigen interactions, such as sandwich immunoassays. We report how a sandwich immunoassay with magnetic particles as label can be used to probe interference by surfactants. Surfactants are often used to improve the performance of immunoassays, however the surfactants can affect the involved proteins and the mechanism of action of surfactant molecules on the antibody-antigen system is mostly unknown. As an example, we investigated molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay with two monoclonal antibodies. The influence of the surfactant below the critical micelle concentration (0.00-0.04%) on dissociation properties was quantified in a magnetic tweezers setup, where a force is applied to the molecules via magnetic particle labels. The force-dependent dissociation curves revealed the existence of two distinct cTn-dependent bond types, namely a weak bond attributable to non-specific binding of cTn, and a strong bond attributable to the specific binding of cTn. The dissociation rate constant of the strong bonds increased with the surfactant concentration by about a factor of two. Circular dichroism spectroscopy data showed that the nonionic surfactant influences the conformation of cTn while not noticeably affecting the two monoclonal antibodies. This suggests that the surfactant-induced increase of the dissociation rate of the specific sandwich-type cTn binding may be related to a conformational change of the antigen molecule. The described methodology is an effective tool to study the influence of surfactants and other interferences on assays based on protein interactions.
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Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Imunoensaio/métodos , Magnetismo , Anticorpos Monoclonais/imunologia , Dicroísmo Circular , Poloxâmero/química , Tensoativos/química , Troponina/imunologiaRESUMO
Magnetic particles are widely used in biological research and bioanalytical applications. As the corresponding tools are progressively being miniaturized and integrated, the understanding of particle dynamics and the control of particles down to the level of single particles become important. Here, we describe a numerical model to simulate the dynamic behavior of ensembles of magnetic particles, taking account of magnetic interparticle interactions, interactions with the liquid medium and solid surfaces, as well as thermal diffusive motion of the particles. The model is verified using experimental data of magnetic field-induced disaggregation of magnetic particle clusters near a physical surface, wherein the magnetic field properties, particle size, cluster size, and cluster geometry were varied. Furthermore, the model clarifies how the cluster configuration, cluster alignment, magnitude of the field gradient, and the field repetition rate play a role in the particle disaggregation process. The simulation model will be very useful for further in silico studies on magnetic particle dynamics in biotechnological tools.
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Coloides/química , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Modelos Químicos , Simulação por Computador , Difusão , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Much has been published about smoking and alcohol intake influencing male fertility, sperm parameters and reproductive outcome. However, there is no conclusive agreement about the effects of cigarette smoking and alcohol use on these outcomes and thus no generally accepted guidelines. The combined effect of cigarette smoking and alcohol intake, though, has not been rigorously investigated. Because alcohol consumption and smoking are often seen together, this study focuses on the effect of smoking and drinking habits separately and combined on semen parameters, such as volume, sperm count, motility and morphology, and on pregnancy outcome. These suggested toxic effects are studied in a group of subfertile, asthenozoospermic men (<10% motile spermatozoa), compared with a group of 'proven fertile', healthy men. The extreme asthenozoospermic group has especially been chosen because of the suspected effect, that is, oxidative stress, on sperm motility. In our study, we found that cigarette smoking and alcohol intake did not differ between the subfertile and fertile group. In conclusion, cigarette smoking and alcohol consumption do not appear to significantly affect sperm parameters, such as volume, sperm count, motility and morphology or pregnancy outcome in our study population.
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Consumo de Bebidas Alcoólicas/efeitos adversos , Infertilidade Masculina/etiologia , Resultado da Gravidez , Fumar/efeitos adversos , Adulto , Astenozoospermia/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , Análise do Sêmen , Motilidade dos EspermatozoidesRESUMO
Platelets get easily activated when in contact with a surface. Therefore in the design of microfluidic blood analysis devices surface activation effects have to be taken into account. So far, platelet-surface interactions have been quantified by morphology changes, membrane marker expression or secretion marker release. In this paper we present a simple and effective method that allows quantification of platelet-surface interactions in real-time. A calcium indicator was used to visualize intracellular calcium variations during platelet adhesion. We designated cells that showed a significant increase in cytosolic calcium as responding cells. The fraction of responding cells upon binding was analyzed for different types of surfaces. Thereafter, the immobilized platelets were chemically stimulated and the fraction of responding cells was analyzed. Furthermore, the time between the binding or chemical stimulation and the increased cytosolic calcium level (i.e. the response delay time) was measured. We used surface coatings relevant for platelet-function testing including Poly-L-lysine (PLL), anti-GPIb and collagen as well as control coatings such as Bovine Serum Albumin (BSA) and mouse immunoglobulin (IgG). We found that a lower percentage of responding cells upon binding, results in a higher percentage of responding cells upon chemical stimulation after binding. The measured delay time between platelet binding under sedimentation and calcium response was the lowest on a PLL-coated surface, followed by an anti-GPIb and collagen-coated surface and IgG-coated surface. The presented method provides real-time information of platelet-surface interactions on a single cell as well as on a cell ensemble level. For future in-vitro diagnostic tests, this real-time single-cell function analysis can reveal heterogeneities in the biological processes of a cell population.
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Plaquetas/metabolismo , Sinalização do Cálcio/fisiologia , Animais , Plaquetas/citologia , Humanos , Camundongos , Microscopia de Fluorescência/métodos , Adesividade PlaquetáriaRESUMO
PURPOSE: To examine the reproducibility of the institutional version of the Dutch Activity Card Sort (ACS-NL) and the possible presence of gender bias. METHODS: Older rehabilitation inpatients (N = 52) were included. Intra- and inter-rater agreement for the ACS-NL total and subscale scores was examined by intraclass correlations (ICC), and agreement of individual items by the κ coefficient (k). Gender bias was examined by the proportion of men and women selecting an ACS item. RESULTS: ICC for inter-rater agreement of the ACS total score ranged between 0.78 and 0.87, ICC for intra-rater agreement ranged between 0.79 and 0.89. Median inter-rater κ for ACS-NL items was 0.72 (interquartile scores; 0.62-0.80). The inter-rater agreement (k = 0.43) and intra-rater agreement (k = 0.39) for the five most important activities was lower. Twenty ACS activities favoured men and seven activities favoured women. As a result, men scored systematically higher on the ACS-NL than women. Logistic regression analysis correcting for activity engagement level confirmed our findings. CONCLUSIONS: The reproducibility of the ACS-NL was high. The ACS-NL institutional version score may be biased in favour of men.
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Atividades Cotidianas , Avaliação da Deficiência , Reabilitação , Sexismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pacientes Internados , Atividades de Lazer , Masculino , Pessoa de Meia-Idade , Países Baixos , Variações Dependentes do Observador , Centros de Reabilitação , Reprodutibilidade dos Testes , Fatores SexuaisRESUMO
We demonstrate the ability to measure torsion stiffness of a protein complex by applying a controlled torque on a magnetic particle. As a model system we use protein G bound to an IgG antibody. The protein pair is held between a magnetic particle and a polystyrene substrate. The angular orientation of the magnetic particle shows an oscillating behavior upon application of a rotating magnetic field. The amplitude of the oscillation increases with a decreasing surface coverage of antibodies on the substrate and with an increasing magnitude of the applied field. For decreasing antibody coverage, the torsion spring constant converges to a minimum value of 1.5 × 10(3) pN·nm/rad that corresponds to a torsion modulus of 4.5 × 10(4) pN·nm(2). This torsion stiffness is an upper limit for the molecular bond between the particle and the surface that is tentatively assigned to a single protein G-IgG protein pair. This assignment is supported by interpreting the measured stiffness with a simple mechanical model that predicts a two orders of magnitude larger stiffness for the protein G-IgG complex than values found for micrometer length dsDNA. This we understand from the structural properties of the molecules, i.e., DNA is a long and flexible chain-like molecule, whereas the antibody-antigen couple is orders of magnitude smaller and more globular in shape due to the folding of the molecules.
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Imunoglobulina G/química , Magnetismo/métodos , Proteínas do Tecido Nervoso/química , Torção Mecânica , Imunoglobulina G/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Tempo , TorqueRESUMO
Biomarkers are gaining increasing interest to predict risk but also to aid in diagnostics. Tissue-specific biomarkers are of utmost importance to detect diseases of respective organs. As of yet there are no atrium-specific biomarkers for risk stratification of atrial disease, such as atrial fibrillation. Bioinformatics such as mRNA microarrays can help to detect tissue-enriched and possibly tissue-specific expressed genes that can be targets for biomarkers. We describe an approach to identify genes preferably expressed in atrial cardiomyocytes compared with ventricular cardiomyocytes by RNA microarray and confirmed by quantitative real-time polymerase chain reaction. By this approach we identified several atrium-enriched genes but also ventricle-enriched genes. As expected atrial natriuretic peptide (ANP) mRNA showed higher expression in atrial cardiomyocytes while with adrenergic stimulation expression was almost as high in ventricular as in atrial cells. Brain-type natriuretic peptide (BNP), however, was not different between atrial and ventricular cells giving a possible explanation for increased levels of NT-proBNP in atrial fibrillation patients. Interesting identified candidates are serpine1 and ltbp2 as atrium-enriched genes whereas alpha-adrenergic receptor subtype 1b and S100A1 expression was significantly higher in ventricular cells. The identified genes need to be confirmed in human tissue and might ultimately be tested as potential biomarkers for atrial stress. (Neth Heart J 2010;18:610-4.).
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Gradients in the concentrations of free phosphate (Pi) and calcium (Ca) exist in fully developed growth zones of long bones and ribs, with the highest concentrations closest to the site of mineralization. As high concentrations of Pi and Ca induce chondrocyte maturation and apoptosis, it has been hypothesized that Ca and Pi drive chondrocyte differentiation in growth plates. This study aimed to determine whether gradients in the important spectral elements phosphorus (P), Ca and sulphur (S) are already present in early stages of development, or whether they gradually develop with maturation of the growth zone. We quantified the concentration profiles of Ca, P, S, chloride and potassium at four different stages of early development of the distal growth plates of the porcine femurs, using particle-induced X-ray emission and forward- and backward-scattering spectrometry with a nuclear microprobe. A Ca concentration gradient towards the mineralized area and a stepwise increase in S was found to develop slowly with tissue maturation. The increase in S co-localizes with the onset of proliferation. A P gradient was not detected in the earliest developmental stages. High Ca levels, which may induce chondrocyte maturation, are present near the mineralization front. As total P concentrations do not correspond with former free Pi measurements, we hypothesize that the increase of free Pi towards the bone-forming site results from enzymatic cleavage of bound phosphate.
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Cálcio/análise , Lâmina de Crescimento/química , Lâmina de Crescimento/embriologia , Fósforo/análise , Enxofre/análise , Suínos/embriologia , Animais , Cloretos/análise , Microanálise por Sonda Eletrônica , Desenvolvimento Embrionário/fisiologia , Idade Gestacional , Potássio/análise , Suínos/metabolismoRESUMO
Observational studies of sitting have shown that, during spontaneous sitting, people adopt a variety of postures. Various researchers have formulated theories to explain why people adopt their sitting postures. Branton (1969) hypothesized that there is continual need for postural stability while sitting. Dempster (1955) stated that additional stability could be obtained through temporarily closing chains of body segments, or, in other words, through decreasing the number of degrees of freedom of the body. The present study elaborates on Dempster's theory. The aim of this study was to determine the influence of the degrees of freedom of the body on postural stability in sitting postures. For 21 different sitting postures, the total number of degrees of freedom was determined. Postural sway, a measure for postural stability, was determined using a 3D motion and position measurement system with ten healthy subjects. This study shows that the mean path length at the level of the second thoracic vertebra (PL0.05), a measure derived from postural sway, increases significantly (p < 0.0001) with an increase of the number of degrees of freedom of the body (DoFB). Closer examination of the data showed that a model taking into account only the degrees of freedom of the lumbar and thoracic spine and pelvis seems to be a better predictor of postural sway than the total number of degrees of freedom of the body.
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Equilíbrio Postural/fisiologia , Postura/fisiologia , Fenômenos Biomecânicos , Feminino , Humanos , Região Lombossacral , Masculino , Movimento , Músculo Esquelético , Suporte de CargaRESUMO
In this paper the adoption of technological innovations to improve the work of bricklayers and bricklayers' assistants is evaluated. Two studies were performed among 323 subjects to determine the adoption of the working methods, the perceived workload, experiences with the working methods, and the reasons for adopting the working methods. Furthermore, a comparison of the results of the studies was made with those of two similar studies in the literature. The results show that more than half of the sector adopted the innovations. The perceived workload was reduced. The employees and employers are satisfied with the working methods and important reasons for adoption were cost/benefit advantages, improvement of work and health, and increase in productivity. Problems preventing the adoption were the use of the working methods at specific sites, for instance in renovation work. The adoption of the new working methods could perhaps have been higher or faster if more attention had been paid to the active participation of bricklayers and bricklayers' assistants during the development of the new working methods and to the use of modern media techniques, such as the Internet and CD/DVD.
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Materiais de Construção , Remoção , Saúde Ocupacional , Desenho de Equipamento , Segurança de Equipamentos , Humanos , Remoção/efeitos adversos , Doenças Profissionais/prevenção & controle , Postura , Estresse Mecânico , Carga de TrabalhoRESUMO
A fast and reproducible production method for pure gaseous [13N]NH3 pulses has been developed. 13N was produced by irradiation of water with 500 nA of 16 MeV protons via the 16O(p,alpha)13N reaction. A mixture of DeVarda's alloy and NaOH was used to convert the produced nitrate/nitrite to ammonia. Pre-treatment of this mixture with water increased the concentrated production of gaseous [13N]NH3. Separation and further purification of ammonia were performed with GC, leading to 5s pulses (1.1 ml STP) with 3.5 +/- 0.5 MBq [13N]NH3 with a high chemical and radiochemical purity suited for positron emission profiling (PEP).
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Enzymically active subcomplexes were purified from bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I) by sucrose-gradient centrifugation in the presence of detergents. These subcomplexes, named I lambda, IS, and I lambda S, catalyse ferricyanide and ubiquinone-1 (Q-1) reduction by NADH at similar rates to complex I, but do not catalyse the reduction of decylubiquinone. In addition, the Q-1 reductase activity of all the subcomplexes is insensitive to rotenone. Chemical and EPR analyses of the subcomplexes show that FMN and all the Fe-S clusters of complex I are present, but that the line shape of cluster 2 is modified. The smallest subcomplex, I lambda S, contains only approximately 13 subunits, as compared to approximately 22 in the previously described subcomplex I alpha [Finel, M., Skehel, J. M., Albracht, S. J. P., Fearnley, I. M. & Walker, J. E. (1992) Biochemistry 31, 11425-11434], but it retains the 75-, 51-, 49-, 30-, 24-, 23- (TYKY) and 20-kDa (PSST) subunits, which are suggested to form a functional core that comprises the EPR-detectable Fe-S clusters 1-4, and FMN. The structural and functional implications of such an arrangement are discussed.
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NADH NADPH Oxirredutases/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Centrifugação , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Complexo I de Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Mononucleotídeo de Flavina/análise , Proteínas Ferro-Enxofre/análise , Mitocôndrias/enzimologia , Dados de Sequência Molecular , NADH NADPH Oxirredutases/químicaRESUMO
The reaction of coupled submitochondrial particles (SMP) with NADH was studied in the absence and presence of the uncoupler gramicidin, both in pre-steady-state and steady-state experiments. It was shown that the formation of ubisemiquinones associated with NADH:Q oxidoreductase is insensitive to uncouplers. It was found, however, that in the absence of gramicidin the ubisemiquinone showed a noticeably faster relaxation than in the presence of this uncoupler. During steady-state oxidation of NADH by coupled submitochondrial particles, the EPR signal of iron-sulphur cluster 2 of complex I, the cluster that is generally believed to be the electron donor for ubiquinone, showed some remarkable changes. Its gz line seemed to disappear from the spectrum, although the gxy line remained clearly present. Detailed EPR analysis indicated that (a component of) the gz line shifted to higher field. The temperature dependence of the EPR signal of cluster 2 was affected as well. In the presence of uncoupler the EPR properties of cluster 2 were indistinguishable from those in particles that showed no intrinsic coupling. These experiments strongly indicate that the coordination of cluster 2 is different in energized and non-energized SMP. The pre-steady-state reaction between these submitochondrial particles and NADH showed that the uncoupler-sensitive changes in both the ubisemiquinone and cluster 2 became effective between 9 ms and 30 ms. Similar changes were observed during succinate-driven reverse electron transfer. This report shows, for the first time, energy-induced structural changes in NADH:Q oxidoreductase.
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Mitocôndrias/enzimologia , NADH NADPH Oxirredutases/química , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons , Radicais Livres , NAD/química , NAD(P)H Desidrogenase (Quinona)/química , OxirreduçãoRESUMO
Until now ubisemiquinones associated with NADH:ubiquinone oxidoreductase (complex I) have been reported to occur in isolated enzyme and in tightly coupled submitochondrial particles. In this report it is shown that ubisemiquinones are always detectable during steady-state electron transfer from NADH to ubiquinone, independent of the type of inner-membrane preparation used. The EPR signal of the rotenone-sensitive ubisemiquinones could be detected not only in coupled MgATP submitochondrial particles, but also in routine preparations of uncoupled submitochondrial particles and in mitochondria. The ubisemiquinone formation in coupled preparations was completely insensitive to uncouplers. The maximal radical concentration during steady-state electron transfer from NADH to quinone was equal to that of iron-sulphur cluster 2. Experiments with antimycin, myxothiazol and 2-thenoyltrifluoroacetone demonstrated that about half of this radical was associated with complex I, giving a ubisemiquinone concentration of about 0.5 mol semiquinone/mol cluster 2. Uncoupled submitochondrial particles, prepared by extensive sonification, never showed radical signals within 100 ms after mixing with NADH. This was due to the reversible inactivation of the enzyme, caused by elevated temperatures during sonification. In preparations with deliberately heat-inactivated complex I, no radical signals were detected within 200 ms after mixing with NADH; at 1 s, however, radical formation was maximal. Yet, depending on the procedure of reactivation of the complex, in preparations previously treated to inactivate them ubisemiquinone concentrations were always less than in untreated particles. When complex I was in the active state the ubisemiquinone signal was maximal within 40 ms. The results described in this report lead to the conclusion that ubisemiquinones form obligatory intermediates in the reaction of NADH dehydrogenase with ubiquinone.