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The atomic beam probe diagnostic concept aims at measuring the edge magnetic field and through that edge current distribution in fusion plasmas by observing trajectories of an ion beam stemming from a diagnostic neutral beam. The diagnostic potentially has microsecond scale time resolution and can thus prove to be a powerful option to study fast changes in the edge plasma. A test detector has been installed on the COMPASS tokamak as an extension of the existing lithium beam diagnostic system. It employs a relatively simple concept of an array of conductive detection plates measuring the incident ion current, which is then amplified and converted to a voltage signal. The aim of the test detector is to experimentally examine the idea of the diagnostic and provide background data for design and installation of a final detector. Also, a numerical code based on the CUDA parallel computing platform has been developed for modeling lithium ion trajectories in the given COMPASS plasma discharges. We present the developments of the detector design and test measurements of the diagnostic performed both in a laboratory beam system and on the COMPASS tokamak.
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This paper describes a new filamentary probe recently introduced on the COMPASS tokamak. It allows the measurement of electrostatic and magnetic properties of the filaments and their changes in dependence on distance from the separatrix in the region between a divertor and midplane. The probe head is mounted on a manipulator moving the probe radially on a shot-to-shot basis. This configuration is suitable for the long term statistical measurement of the plasma filaments and the measurement of their evolution during their propagation from the separatrix to the wall. The basics of the filamentary probe construction, the evolution of the plasma parameters, and first conditional averages of the plasma filaments in the scrape-off layer of the COMPASS tokamak during the L-mode regime are presented.
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The ball-pen probe (BPP) technique is used successfully to make profile measurements of the electron temperature on the ASDEX Upgrade (Axially Symmetric Divertor Experiment), COMPASS (COMPact ASSembly), and ISTTOK (Instituto Superior Tecnico TOKamak) tokamak. The electron temperature is provided by a combination of the BPP potential (ΦBPP) and the floating potential (Vfl) of the Langmuir probe (LP), which is compared with the Thomson scattering diagnostic on ASDEX Upgrade and COMPASS. Excellent agreement between the two diagnostics is obtained for circular and diverted plasmas and different heating mechanisms (Ohmic, NBI, ECRH) in deuterium discharges with the same formula Te = (ΦBPP - Vfl)/2.2. The comparative measurements of the electron temperature using BPP/LP and triple probe (TP) techniques on the ISTTOK tokamak show good agreement of averaged values only inside the separatrix. It was also found that the TP provides the electron temperature with significantly higher standard deviation than BPP/LP. However, the resulting values of both techniques are well in the phase with the maximum of cross-correlation function being 0.8.
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A modified Katsumata probe has been developed for measurement of ion velocity distribution function (IVDF) in technological non-magnetized plasmas. A simple construction of the modified Katsumata probe consists of adding a pair of permanent Sm-Co magnets in front of Katsumata probe. A comparative study regarding IVDF measurement in a high power impulse magnetron sputtering system operating in pure argon atmosphere by means of developed modified Katsumata probe and commercially available gridded retarding field analyzer (RFA) has been carried out. A time-resolved measurement of IVDF for two different pressures whilst other plasma conditions have been kept unchanged has revealed that the main advantage of the modified Katsumata probe compared to the RFA consists in significantly smaller angular aperture of entrance orifice of modified Katsumata probe being approximately 15° in comparison with a commercial RFA having angular aperture more than 160°. It leads in much better velocity resolution in measured IVDF since the transversal part of velocity vector is much more suppressed compared to RFA. Furthermore, the modified Katsumata probe less suffers from collisions of ions in the space charge sheath in front or inside of the probe compared to the RFA.
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Inactivity and more sedentary time predominate the daily activity level of many of today's children. In Denmark, certified sport after-school cares have been established in order to increase children's daily physical activity (PA) level. This cross-sectional study aimed to investigate the activity level among participants in certified sport after-school cares vs regular after-school cares. The study was carried out in 2011 in 10 after-school cares (5 sport/5 regular) throughout Denmark, whereof 475 children aged 5-11 years participated. PA level was assessed using Actigraph GT3X and GT3X+ activity monitors worn by the children for at least 8 consecutive days. Anthropometry and cardiorespiratory fitness were measured as well. A multivariate regression analysis was carried out to check for the differences in the PA level across the two care systems. However, there did not appear to be any differences in overall PA or in time-specific day parts (e.g., during after-school care). The activity levels were quite similar across after-school cares and were mutually high during time spent in the care facility.
Assuntos
Certificação , Promoção da Saúde , Atividade Motora/fisiologia , Serviços de Saúde Escolar/organização & administração , Esportes/normas , Actigrafia , Estatura , Água Corporal , Criança , Pré-Escolar , Estudos Transversais , Dinamarca , Teste de Esforço , Feminino , Humanos , Masculino , Esforço Físico , Aptidão Física , Circunferência da CinturaRESUMO
PURPOSE: To evaluate an irregular perilesional hyperechoic zone as a potential criterion of malignancy on breast ultrasound and to test whether this zone correlates with perilesional T2 hyperintensity on magnetic resonance imaging (MRI). MATERIALS AND METHODS: A total of 137 patients (85 malignant lesions, 52 benign lesions) who underwent breast ultrasound with a 9â-â14 MHz linear broad-spectrum transducer and consecutive ultrasound-guided biopsy were included. All patients additionally underwent breast MR imaging with dedicated breast coils at 1.5 T. The protocol included a T2-weighted sequence. Perilesional hyperechoic and T2 hyperintense areas were measured by planimetry using the slice showing maximum extension of this area. The sensitivity and specificity of the perifocal area for identifying breast malignancy were determined using ROC analysis. Correlation was assessed using Pearson analysis. RESULTS: The presence of a hyperechoic zone identified malignancy with a sensitivity of 87% and a specificity of 81%. Additionally, there was a highly significant correlation of the size of the hyperechoic zone with the degree of tumor differentiation (pâ=â0.002) as well as with the mib-1 proliferation index (pâ=â0.006) and lymphangio-invasion (pâ=â0.02). No significant correlation was found between the hyperchoic zone and the T2 hyperintense zone on MRI (R2â=â0.16). CONCLUSION: A hyperechoic zone surrounding breast lesions may serve as an additional sonomorphologic criterion of malignancy. Yet, it does not seem to correlate with edema on MRI.
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Biópsia com Agulha de Grande Calibre , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/diagnóstico por imagem , Carcinoma Ductal de Mama/patologia , Ultrassonografia de Intervenção , Ultrassonografia Mamária/métodos , Adolescente , Adulto , Idoso , Mama/patologia , Proliferação de Células , Diagnóstico Diferencial , Feminino , Humanos , Antígeno Ki-67/análise , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
The core Thomson scattering diagnostic (TS) on the COMPASS tokamak was put in operation and reported earlier. Implementation of edge TS, with spatial resolution along the laser beam up to â¼1/100 of the tokamak minor radius, is presented now. The procedure for spatial calibration and alignment of both core and edge systems is described. Several further upgrades of the TS system, like a triggering unit and piezo motor driven vacuum window shutter, are introduced as well. The edge TS system, together with the core TS, is now in routine operation and provides electron temperature and density profiles.
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Typical situations, which can be met during the process of absolute calibration, are shown in the case of a visible light observation system for the COMPASS tokamak. Technical issues and experimental limitations of absolute measurements connected with tokamak operation are discussed.
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The electron density and temperature profiles measured by the Thomson scattering diagnostic on the COMPASS tokamak are used for estimation of electron kinetic energy, energy confinement time, and effective charge number Z(eff). Data are compared with the line-integrated electron density measured by a microwave interferometer in an ohmically heated plasma with a circular cross section. An error analysis of both electron temperature and density are performed by two methods-a constant chi-square boundaries method and a Monte Carlo simulation, determining asymmetrical error bars for the electron temperature.
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Despite the well-documented health effects of physical activity, few studies focus on the correlates of leisure-time sports and exercise participation. The present study examined correlations between adolescent sports participation and demographic factors, socioeconomic status (SES) and sociocultural factors. A school-based cross-sectional cluster sample including 6356 Danish fifth- and ninth-grade adolescents from four municipalities were included. Age (younger) and gender (boy) were associated with adolescents' sports participation. Girls were half as likely [odds ratio (OR) 0.49 95% confidence interval (CI): 0.44-0.55] to participate in sports than boys. Adolescents were more likely to participate in sports if they perceived their parents as active in exercise or sports. Adolescents with one or two unemployed parents were 0.75 (95% CI: 0.62-0.89) and 0.75 (95% CI: 0.56-1.00), respectively, less likely to participate in sports than adolescents with two employed parents. In a gender-stratified analysis, parents' occupational status was only a predictor of sports participation in girls. Differences between municipalities in adolescents' sports participation remained significant when controlled for individual factors such as gender, age, parents' background or parents' physical activity. The association between sociocultural and SES was stronger for girls than boys. In conclusion, demographics, SES and sociocultural factors were the best determinants of adolescent sport participation.
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Cultura , Relações Pais-Filho , Classe Social , Esportes , Adolescente , Criança , Intervalos de Confiança , Estudos Transversais , Dinamarca , Exercício Físico , Feminino , Humanos , Atividades de Lazer , Masculino , Razão de ChancesRESUMO
A new Thomson scattering diagnostic system has been designed and is being built now on the COMPASS tokamak at the Institute of Plasma Physics ASCR in Prague (IPP Prague) in the Czech Republic. This contribution focuses on design, development, and installation of the light collection and detection system. High spatial resolution of 3 mm will be achieved by a combination of design of collection optics and connected polychromators. Imaging characteristics of both core and edge plasma collection objectives are described and fiber backplane design is presented. Several calibration procedures are discussed. The operational deployment of the Thomson scattering diagnostic is planned by the end of 2010.
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In Arabidopsis thaliana, the Toc34 receptor component of the chloroplast import machinery is encoded by two independent but highly homologous genes, atToc33 and atToc34. We have isolated a T-DNA insertion mutant of atToc33 which is characterized by a pale phenotype, due to reductions in the levels of photosynthetic pigments, and alterations in protein composition. The latter involve not only chloroplast proteins but also some cytosolic polypeptides, including 14-3-3 proteins which, among other functions, have been proposed to be cytosolic targeting factors for nucleus-encoded chloroplast proteins. Within the chloroplast, many, though not all, proteins of the photosynthetic apparatus, as well as proteins not directly involved in photosynthesis, are found in significantly reduced amounts in the mutant. However, the accumulation of other chloroplast proteins is unaffected. This suggests that the atToc33 receptor is responsible for the import of a specific subset of nucleus-encoded chloroplast proteins. Supporting evidence for this conclusion was obtained by antisense repression of the atToc34 gene in the atToc33 mutant, which results in an exacerbation of the phenotype.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/genética , DNA Bacteriano/genética , Proteínas de Membrana/genética , Mutação/genética , Northern Blotting , Southern Blotting , Western Blotting , Carotenoides/metabolismo , Clorofila/metabolismo , Cloroplastos/ultraestrutura , Clonagem Molecular , Primers do DNA , Fluorescência , Componentes do Gene , Vetores Genéticos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Virgin females of Lobesia botrana Denis & Schiffermüller were mated at ages of 1, 3, 5, 8, 12 and 16 days and the effect of mating delay on reproductive output assessed. Delayed mating did not affect female mating success but fertilization was reduced in 16-day-old females. Increased mating delays substantially affected daily oviposition pattern and resulted in a significant reduction of both fecundity and fertility, so that mean number of viable eggs laid decreased from 150 to 22 in 1- and 16-day-old mated females, respectively. Heavier females were more constrained than lighter ones by mating delays and female reproductive efficiency (no. viable eggs/female body weight) was also significantly reduced. Female longevity significantly increased and oviposition period gradually declined with mating delay. The number of viable eggs was positively correlated with both female weight and oviposition period; female longevity and female weight were also significantly correlated. However, the significance of these correlations declined with increased mating delay. Results overall indicated that mating delay drastically reduces female L. botrana reproductive output. The implications of delayed mating of females are discussed from an ecological perspective in relation to L. botrana control using mating disruption.
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Mariposas/fisiologia , Comportamento Sexual Animal/fisiologia , Animais , Feminino , Fertilidade , Longevidade , Masculino , Oviposição/fisiologia , Reprodução/fisiologia , VitisRESUMO
The infectious prion protein, PrP(Sc), a predominantly beta-sheet aggregate, is derived from PrP(C), the largely alpha-helical cellular isoform of PrP. Conformational conversion of PrP(C) into PrP(Sc) has been suggested to involve a chaperone-like factor. Here we report that the bacterial chaperonin GroEL, a close homolog of eukaryotic Hsp60, can catalyze the aggregation of chemically denatured and of folded, recombinant PrP in a model reaction in vitro. Aggregates form upon ATP-dependent release of PrP from chaperonin and have certain properties of PrP(Sc), including a high beta-sheet content, the ability to bind the dye Congo red, detergent-insolubility and increased protease-resistance. A conserved sequence segment of PrP (residues 90-121), critical for PrP(Sc) generation in vivo, is also required for chaperonin-mediated aggregate formation in vitro. Initial binding of refolded, alpha-helical PrP to chaperonin is mediated by the unstructured N-terminal segment of PrP (residues 23-121) and is followed by a rearrangement of the globular PrP core-domain. These results show that chaperonins of the Hsp60 class can, in principle, mediate PrP aggregation de novo, i.e. independently of a pre-existent PrP(Sc) template.
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Chaperoninas/metabolismo , Príons/química , Príons/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Chaperonina 10/química , Chaperonina 10/metabolismo , Chaperonina 60/química , Chaperonina 60/metabolismo , Chaperoninas/química , Vermelho Congo/metabolismo , Sequência Conservada , Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Modelos Biológicos , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Renaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
Chromate (Cr) decreases the growth of Spirodela polyrhiza. The fronds lost their pigments. The O2 evolution was also decreased. The Cr effect was found to be dose dependent. The toxic effects of Cr have further been studied on the photosynthetic activity of Spirodela polyrhiza by means of the chlorophyll a (Chl a) fluorescence transient O-J-I-P. The Chl a fluorescence transients were recorded in vivo with high time resolution and analyzed according to the JIP-test which can quantify the photosystem II behavior. Cr treated plants show a decrease in yield for primary photochemistry, phi Po. The performance index of PSII, PIABS, which is the combination of the indexes of three independent parameters, (1) the total number of active reaction centers per absorption (RC/ABS), (2) yield of primary photochemistry (phi Po) and (3) efficiency with which a trapped exciton can move an electron into the electron transport chain (psi 0), decreased due to Cr treatment. Chromate sensitivity varies within plant populations. In summary Cr affects several targets of PSII. More specifically, the main targets of Cr, according to the JIP-test, can be listed as a decrease in the number of active reaction centers and damage to the oxygen-evolving complex.
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Araceae/fisiologia , Clorofila/análise , Cromatos/efeitos adversos , Fotossíntese/efeitos dos fármacos , Poluentes Químicos da Água/efeitos adversos , Biomarcadores/análise , Clorofila A , Fluorescência , Oxigênio/metabolismoRESUMO
The infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear. Using recombinant Syrian hamster prion protein [SHaPrP(29-231)], we investigated metal ions as possible ligands of PrP. Near-UV circular dichroism spectroscopy (CD) indicates that the conformation of SHaPrP(29-231) resembles PrPC purified from hamster brain. Here we demonstrate by CD and tryptophan (Trp) fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231). Binding of copper quenches the Trp fluorescence emission significantly, shifts the emission spectrum to shorter wavelengths, and also induces changes in the near-UV CD spectrum of SHaPrP(29-231). The binding sites are highly specific for Cu2+, as indicated by the lack of a change in Trp fluorescence emission with Ca2+, Co2+, Mg2+, Mn2+, Ni2+, and Zn2+. Binding of Cu2+ also promotes the conformational shift from a predominantly alpha-helical to a beta-sheet structure. Equilibrium dialysis experiments indicate a binding stoichiometry of approximately 2 copper molecules per PrP molecule at physiologically relevant concentrations, and pH titration of Cu2+ binding suggests a role for histidine as a chelating ligand. NMR spectroscopy has recently demonstrated that the octarepeats (PHGGGWGQ) in SHaPrP(29-231) lack secondary or tertiary structure in the absence of Cu2+. Our results suggest that each Cu2+ binds to a structure defined by two octarepeats (PHGGGWGQ) with one histidine and perhaps one glycine carbonyl chelating the ion. We propose that the binding of two copper ions to four octarepeats induces a more defined structure to this region.
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Cobre/química , Príons/química , Animais , Sítios de Ligação , Cátions Bivalentes , Dicroísmo Circular , Cobre/metabolismo , Cricetinae , Concentração de Íons de Hidrogênio , Mesocricetus , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Príons/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequências Repetitivas de Ácido NucleicoRESUMO
We investigated the mechanism of refolding and reassembly of recombinant alpha and beta chains of the class II major histocompatibility molecules (MHC-II) HLA-DRB5*0101. Both chains were expressed in the cytosol of Escherichia coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the alpha and beta chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short-chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.
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Antígenos HLA-DR/química , Conformação Proteica , Dobramento de Proteína , Dicroísmo Circular , Detergentes/farmacologia , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Polarização de Fluorescência , Antígenos HLA-DR/genética , Cadeias HLA-DRB5 , Humanos , Ligantes , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , TriptofanoRESUMO
PURPOSE: The purpose of this study was to determine the response rate of paclitaxel administered at maximal tolerated doses (MTD) in patients with newly diagnosed glioblastoma multiform. PATIENTS AND METHODS: All patients in this multicenter study were 45 years or older and had measurable residual tumor on postoperative MRI scans. Up to 3 cycles of paclitaxel were administered as a continuous 96-hour intravenous infusion prior to radiation, provided that the tumor did not enlarge on serial MRIs. The initial 10 patients were treated with the previously recommended phase II dose of 140 mg/m2. Less than anticipated toxicity led to the development of a phase I/II study in 24 patients in which paclitaxel doses were escalated separately in patients receiving (+EIAED) or not receiving (-EIAED), concomitant enzyme-inducing antiepileptic drugs. Paclitaxel plasma steady-state concentrations (Css) were measured during the first cycle of chemotherapy. Response was the primary efficacy endpoint for this study, although survival was also assessed. RESULTS: The MTD was 140 mg/m2 in the -EIAED, and 200 mg/m2 in the +EIAED patient groups. The mean Css for the -EIAED patients treated at 140 mg/m2 was 38 nM, whereas the mean Css for +EIAED patients were 17 nm at 140 mg/m2, 27 nM at 175 mg/m2, 46 nM at 200 mg/m2, and 51 nM at 230 mg/m2. One patient, who had a verified partial response, had his diagnosis changed to an anaplastic oligodendroglioma on subsequent central neuropathologic review. None of the 15 assessable glioblastoma patients treated at or above the MTD doses showed a radiographic response to paclitaxel. The median survival of eligible patients on this protocol was 355 days (95% CI, 255 to 485 days), which is similar to the survival of comparable patients treated with conventional therapy. CONCLUSIONS: These results suggest that (1) paclitaxel given as a 96-hour infusion at the MTD has minimal activity in patients with untreated glioblastoma, (2) the concomitant administration of EIAEDs alters the pharmacology of paclitaxel, resulting in a lower Css, reduced systemic toxicity, and higher dose requirements, (3) this study design, in which a new agent is given prior to radiation therapy (with serial monitoring of MRI), did not adversely affect survival in this patient population.
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Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Paclitaxel/uso terapêutico , Idoso , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/sangue , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/cirurgia , Quimioterapia Adjuvante , Interações Medicamentosas , Feminino , Glioblastoma/radioterapia , Glioblastoma/cirurgia , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Paclitaxel/efeitos adversos , Paclitaxel/sangue , Radioterapia Adjuvante , Análise de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.
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Príons/química , Proteínas Recombinantes/química , Animais , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Cricetinae , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mesocricetus , Maleabilidade , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , UltracentrifugaçãoRESUMO
The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.