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INTRODUCTION: Hyporesponsiveness to erythropoiesis-stimulating agents (ESAs) affects 10-15% of the chronic dialysis population. We explored baseline characteristics and predictors of ESA hyporesponsiveness in a global randomized cardiovascular outcomes study comparing an investigational hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI), daprodustat, with conventional ESA treatment. METHODS: ASCEND-D (NCT02879305) recruited 2,964 chronic dialysis patients receiving ESA treatment (standardized to weekly intravenous [IV] epoetin) who were iron replete at baseline. The primary ESA hyporesponsiveness definition was an ESA Resistance Index (ERI, ESA units/kg/week/hemoglobin g/L) ≥2 or IV standardized ESA dose ≥450 units/kg/week. Predictors of ESA hyporesponsiveness were determined using a multivariable regression model. Alternative hyporesponder definitions were explored. RESULTS: Using the primary definition, 354 (12%) patients were ESA hyporesponsive. Geographic region, notably Latin America, lower baseline body mass index and transferrin saturation, younger age, lower albumin concentration, and a higher baseline IV iron dose were identified as strongly associated (p < 0.001) with ESA hyporesponsiveness. Additional predictors of ESA hyporesponsiveness included female sex (p = 0.010), history of heart failure (p = 0.035), longer dialysis vintage (p = 0.077), smoking status (p = 0.247), aspirin use (p = 0.121), and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker use (p = 0.214). CONCLUSION: This is the first global HIF-PHI study to report prespecified definitions and predictors of ESA hyporesponsiveness. While most of the predictors identified in our study have been previously reported, geographic region stands out as an unexpected finding, meriting further investigation.
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Anemia , Hematínicos , Humanos , Feminino , Hematínicos/uso terapêutico , Hematínicos/farmacologia , Diálise Renal/efeitos adversos , Anemia/tratamento farmacológico , Eritropoese , Hemoglobinas , Ferro/uso terapêuticoRESUMO
AIM: Preeclampsia and obesity are two closely related syndromes. The high maternal prepregnancy body mass index (BMI) is a risk factor for present preeclampsia, independently of the ethnic background of the studied population. The aim of this study was to analyse in a prospective cohort study the relation between prepregnancy BMI and development of preeclampsia in Maya-Mestizo women. DESIGN: This is a prospective cohort study of 642 pregnant women that were included in the first trimester of the pregnancy (gestational age ≤12 weeks at the first antenatal visit) and all of them were of Maya-Mestizo ethnic origin from the state of Yucatán, México. We assessed the potential risk factors for preeclampsia and documented the prepregnancy BMI (kg/m2) that was based on measured height and maternal self-report of prepregnancy weight at the initial visit. Besides, in the antenatal visit we documented if the pregnant women developed preeclampsia. RESULTS: Of the 642 pregnant Maya-Mestizo women, 49 developed preeclampsia, with an incidence of 7.6% (44.9% had severe and 55% mild). The prepregnancy BMI was higher in women with developed preeclampsia than in those with normal pregnancies. Women with overweight or obesity in comparison with normal weight presented a RR = 2.82 (95% CI: 1.32-6.03; P = 0.008) and RR= 4.22 (95% CI: 2.07-8.61; P = 0.001), respectively. CONCLUSIONS: Our findings expand the previous studies to show that the higher prepregnancy BMI is a strong, independent risk factor for preeclampsia.
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Índice de Massa Corporal , Obesidade/epidemiologia , Pré-Eclâmpsia/epidemiologia , Adulto , Feminino , Humanos , Incidência , México/epidemiologia , Gravidez , Estudos Prospectivos , Fatores de Risco , Adulto JovemRESUMO
The aim of this study was to isolate, identify and quantify soluble free phenolics, conjugated acid-hydrolysable phenolics (AHP) and alkaline-hydrolysable phenolics, and bound phenolics (BP) fractions from two tomato varieties (saladette and grape) and an industrial tomato by-product, as well as, to determine their antioxidant capacity. Phenolic composition was determined using Folin-Ciocalteu's method and HPLC-DAD. AHP were predominant in grape and saladette tomato extracts (91.47 ± 17.28 mg gallic acid equivalents (GAE) per g dry extract (DE) and 57.41 ± 8.80 mg GAE per g DE, respectively), while BP form was predominant in tomato by-product (51.30 ± 10.91 GAE per g DE). AHP extract of grape tomato presented the highest antioxidant capacity by DPPH assay (252.35 ± 42.55 µmol trolox equiv (TE) per g DE). In the case of ORAC assay, AHP fractions from both grape (1005.19 ± 138.52 µmol TE per g DE) and saladette tomatoes (804.16 ± 131.45 µmol TE per g DE), and BP fraction from by-product (852.40 ± 71.46 µmol TE per g DE) showed the highest ORAC values. Caffeic acid was the most abundant phenolic acid and it was found mainly in its conjugated forms. Naringenin was the most abundant flavonoid and it was mainly detected in bound form. Our analysis allowed a better characterization of phenolic compounds in whole tomato and by-product, remarking the importance of the fractionation. The valorization of the industrial tomato by-product, through the use of its different fractions of phenolic antioxidant compounds, could generate additional income to the tomato industry and reduce the waste disposal problem.
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Bioactive compounds and antioxidant activity were evaluated from industrial Jalapeño pepper byproducts and simulated non processed byproducts from two Mexican states (Chihuahua and Sinaloa) to determine their value added potential as commercial food ingredients. Aqueous 80% ethanol produced about 13% of dry extract of polar compounds. Total phenolic content increased and capsaicin and dihydrocapsaicin decreased on scalding samples (80 °C, 2 min) without affecting ascorbic acid. The major phenolic compounds, rutin, epicatechin and catechin comprised 90% of the total compounds detected by HPLC of each Jalapeño pepper byproducts. ORAC analysis showed that the origin and scalding process affected the antioxidant activity which correlated strongly with capsaicin content. Although scalding decreased capsaicinoids (up to 42%), phenolic content by (up to 16%), and the antioxidant activity (variable). Jalapeño pepper byproduct is a good source of compounds with antioxidant activity, and still an attractive ingredient to develop useful innovative products with potential food/non-food applications simultaneously reducing food loss and waste.
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The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6 U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39 U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70 kDa in C. cladosporioides and ~ 21, 63 and 100 kDa in P. funiculosum. The best results for saccharification (10.11 g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36 h of the hydrolytic process at pH 5 and 50 °C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.
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BACKGROUND: The potential use of hard-to-cook (hardened) chickpeas to obtain value-added functional food ingredients was evaluated. For that purpose, some nutraceutical and functional attributes of several chickpea protein hydrolysates (CPHs) prepared from both fresh and hard-to-cook grains were evaluated. RESULTS: All the CPHs prepared from both fresh and hard-to-cook grains, with the enzymes alcalase, pancreatin and papain, showed high angiotensin converting enzyme inhibitory (ACE-I) activity with IC50 values ranging from 0.101 to 37.33 µg mL⻹; similarly, high levels of antioxidant activity (around 18.17-95.61 µmol Trolox equivalent antioxidant capacity µg⻹ CPH) were obtained through both the 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) methods. Regarding functional characterization of the CPHs, oil absorption values ranged from 1.91 to 2.20 mL oil g⻹ CPH, with water solubility almost 100% from pH 7 to 10. CONCLUSION: The high antioxidant and ACE-I activities as well as the good functional properties of the CPH prepared from both fresh and hard-to-cook grains, suggest its use in food formulations with value added in human health.
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Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antioxidantes/farmacologia , Cicer/química , Proteínas Alimentares/farmacologia , Peptidil Dipeptidase A/metabolismo , Hidrolisados de Proteína/farmacologia , Sementes/química , Benzotiazóis , Compostos de Bifenilo/metabolismo , Culinária , Proteínas Alimentares/análise , Suplementos Nutricionais , Enzimas/metabolismo , Alimento Funcional , Humanos , Picratos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Hidrolisados de Proteína/química , Ácidos Sulfônicos/metabolismo , Tiazóis/metabolismoRESUMO
In spite of the negative effects of anger, coaches are often seen becoming angry during games. This is especially worrying in U18 categories. Thus, the objective of this study was to identify the influence that the coach's anger has on the performance of a basketball team in competition. For this, an ad hoc observation tool was designed, in which 587 moments of anger from the coaching staff (64 coaches) were recorded in the 24 semi-final and final matches of the Spanish Autonomous Region Team Championships in 2019 and 2020 in the infantil (M = 14 years old) and cadete (M = 16 years old) categories. The results show that, in response to most incidents of coach anger, the performance of the team did not change. Significant differences were identified in some scenarios, with low- or medium-intensity anger targeted at the defence, where the team performance improved. However, anger towards the referee in the last quarter with scores level had a negative influence on the team's performance.
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Basquetebol , Tutoria , Adolescente , Ira , Ansiedade , HumanosRESUMO
Using molecular dynamics we study the behavior of a large particle immersed in a bed of smaller ones. The system is bidimensional, consisting of many rough inelastic hard disks of equal size plus a larger one: the intruder. All possible parameters of the system are kept fixed except for two dimensionless parameters determining the frequency and amplitude of the vibrating base. A systematic exploration of this parameter space leads to determining a transition line separating a zone in which the Brazil nut effect is observed and one in which it is not. The results strongly suggest that, in the region of the parameter space in which the study is made, there is a minimum amplitude and a maximum frequency for the Brazil nut effect to take place. These results compare well with isolated results from other authors.
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Jatropha curcas seed shells are the by-product obtained during oil extraction process. Recently, its chemical composition has gained attention since its potential applications. The aim of this study was to identify phenolic compounds profile from a non-toxic J. curcas shell from Mexico, besides, evaluate J. curcas shell methanolic extract (JcSME) antioxidant activity. Free, conjugate and bound phenolics were fractionated and quantified (606.7, 193.32 and 909.59 µg/g shell, respectively) and 13 individual phenolic compounds were detected by HPLC. The radical-scavenging activity of JcSME was similar to Trolox and ascorbic acid by DPPH assay while by ABTS assay it was similar to BHT. Effective antioxidant capacity by ORAC was found (426.44 ± 53.39 µmol Trolox equivalents/g shell). The Mexican non-toxic J. curcas shell is rich in phenolic compounds with high antioxidant activity; hence, it could be considerate as a good source of natural antioxidants.
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Antioxidantes/farmacologia , Jatropha/química , Fenóis/análise , Extratos Vegetais/análise , Extratos Vegetais/farmacologiaRESUMO
Amarantin, an 11S globulin, is one of the most important storage proteins of amaranth seeds, with relevant nutritional-functional and nutraceutical characteristics. Its cDNA was cloned in-frame with a sequence encoding a polyhistidine tag and expressed under the direction of a 35S promoter in transgenic tobacco seeds. The presence of a (His)(6) tag on the polypeptide permitted a high-yield single-step purification using immobilized metal-ion affinity chromatography and rapid characterization. Purified His-tag amarantin accounted for up to 5% of total soluble seed protein. Biochemical characterization indicated that purified His-tag amarantin migrated with the expected molecular weight (53 kDa) and was correctly processed into an acidic polypeptide (32 kDa) with isoelectric point (pI) of 5.58 and a basic polypeptide (21 kDa) with pI of 9.24, linked by a disulfide bridge. Moreover, His-tag amarantin was assembled into both homo- and hetero-hexameric 11S structures. These results show that the His tag did not change the biochemical and physicochemical properties of amarantin. The strategy presented here for rapid and high-yield expression and purification procedure should facilitate structure-function studies for this nutritional protein.
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Globulinas/química , Histidina/química , Histidina/isolamento & purificação , Nicotiana/genética , Nicotiana/metabolismo , Proteínas de Plantas/química , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar , Dissulfetos/química , Eletroforese em Gel Bidimensional , Globulinas/genética , Histidina/genética , Ponto Isoelétrico , Peso Molecular , Peptídeos/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Engenharia de Proteínas/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sementes/química , Sementes/genética , UltracentrifugaçãoRESUMO
The effect of friction in the thermal convection instability of granular fluids is studied by means of molecular dynamics simulations. It is found that the transitions between different convective states (zero, one, and two rolls) are primarily governed by the average energy loss per collisions and not by the friction and restitution coefficients separately, and can be roughly described in terms of a single effective restitution coefficient. The average energy loss per collisions, for a fixed value of the restitution coefficient, shows a maximum for a friction coefficient kappa approximately 0.3. The presence of this maximum manifests itself as a reentrant behavior in the transition lines in parameter space when the value of the friction coefficient is increased beyond 0.3.
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DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.
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Amaranthus/genética , Amaranthus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Alérgenos , Antígenos de Plantas , Clonagem Molecular/métodos , Escherichia coli/classificação , Histidina/química , Histidina/genética , Peso Molecular , Proteínas de Plantas/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Armazenamento de Sementes , Sementes/genética , Sementes/metabolismo , Solubilidade , Especificidade da EspécieRESUMO
Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult to perform. Thus, the objective of this work was to assess the food safety of genetically modified maize with an amaranth globulin protein termed amarantin. Transgenic maize lines evaluated showed, in relation to nontransgenic, 4-35% more protein and 0-44% higher contents of specific essential amino acids. Individual sequence analysis with known amino acid sequences, reported as allergens, showed that none of these IgE elicitors were identified in amarantin. Amarantin was digested within the first 15 min by Simulated Gastric Fluid treatment as observed by Western blot. Expressed amarantin did not induce important levels of specific IgE antibodies in BALB/c mice, as analyzed by ELISA. We conclude that the transgenic maize with amarantin is not an important allergenicity inducer, just as nontransgenic maize.
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Alérgenos/imunologia , Amaranthus/química , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas/imunologia , Zea mays/genética , Zea mays/imunologia , Alérgenos/administração & dosagem , Sequência de Aminoácidos , Animais , Digestão , Imunoglobulina E/análise , Imunoglobulina E/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Homologia de SequênciaRESUMO
The purpose of this study was to identify the basketball game-related statistics that best discriminate performances by sex of players and level of competition. Archival data were obtained from the International Basketball Federation boxscores for all games during men's senior (n=62), men's junior (n=64), women's senior (n=62), and women's junior (n=42) World Championships. The game-related statistics gathered included 2- and 3-point field-goals (both successful and unsuccessful), free-throws (both successful and unsuccessful), defensive and offensive rebounds, blocks, assists, fouls, steals and turnovers. For the analysis only the close games were selected (N= 105, 1 to 12 points difference). Men's teams were discriminated from women's teams by their higher percentage of blocks and lower percentage of steals and unsuccessful 2-point field goals. Junior teams were discriminated from senior teams by their lower percentage of assists and higher percentage of turnovers. In the two-factor interaction, the teams were mainly discriminated by the game-related statistics identified for level of competition.
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Basquetebol/estatística & dados numéricos , Comportamento Competitivo , Preconceito , Feminino , Humanos , Masculino , Fatores SexuaisRESUMO
The phenolic content and antioxidant and antimutagenic activities from the peel and seeds of different tomato types (grape, cherry, bola and saladette type), and simulated tomato industrial byproducts, were studied. Methanolic extracts were used to quantify total phenolic content, groups of phenolic compounds, antioxidant activities, and the profile of phenolic compounds (by HPLC-DAD). Antimutagenic activity was determined by Salmonella typhimurium assay. The total phenolic content and antioxidant activity of tomato and tomato byproducts were comparable or superior to those previously reported for whole fruit and tomato pomace. Phenolic compounds with important biological activities, such as caffeic acid, ferulic acid, chlorogenic acids, quercetin-3-ß-O-glycoside, and quercetin, were quantified. Differences in all phenolic determinations due to tomato type and part of the fruit analyzed were observed, peel from grape type showing the best results. Positive antimutagenic results were observed in all samples. All evaluated materials could be used as a source of potential nutraceutical compounds.
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Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Solanum lycopersicum/química , Antimutagênicos/química , Antioxidantes/análise , Cromatografia Líquida de Alta Pressão , Frutas/química , Mutação/efeitos dos fármacos , Fenóis/análise , Extratos Vegetais/análise , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Sementes/química , Resíduos/análiseRESUMO
The arbuscular mycorrhizal (AM) symbiosis is an intimate association between specific soil-borne fungi and the roots of most land plants. AM colonisation elicits an enhanced defence resistance against pathogens, known as mycorrhizal-induced resistance (MIR). This mechanism locally and systemically sensitises plant tissues to boost their basal defence response. Although a role for oxylipins in MIR has been proposed, it has not yet been experimentally confirmed. In this study, when the common bean (Phaseolus vulgaris L.) lipoxygenase PvLOX2 was silenced in roots of composite plants, leaves of silenced plants lost their capacity to exhibit MIR against the foliar pathogen Sclerotinia sclerotiorum, even though they were colonised normally. PvLOX6, a LOX gene family member, is involved in JA biosynthesis in the common bean. Downregulation of PvLOX2 and PvLOX6 in leaves of PvLOX2 root-silenced plants coincides with the loss of MIR, suggesting that these genes could be involved in the onset and spreading of the mycorrhiza-induced defence response.
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Amarantin is the predominant seed storage protein from amaranth. It shows a high content of essential amino acids, making this protein important from a nutritional viewpoint. The protein has two disulfide linked subunits: acidic and basic. Acidic subunit has the potential as a functional and nutraceutical protein, and it is structurally a good candidate for modification. In order to improve its functionality, the primary structure was modified in the third variable region of globulins 11S, by inserting four Val-Tyr antihypertensive peptides in tandem. The designed plasmid was expressed in Escherichia coli Origami (DE3) and then the expressed protein was purified. Mass spectrometry analysis was used to corroborate the identity of the protein by peptide mass fingerprinting; also, the modified peptide was fragmented and sequenced by mass spectrometry, corroborating thus the inserted residues. The hydrolyzed protein showed a high inhibitory activity of the angiotensin converting enzyme (IC(50) 0.064 mg ml(-1)); it was nearly eightfold more active than the nonmodified protein. In spite that the nonmodified subunit is less active, its activity is comparable with other hydrolyzed proteins reported as high active inhibitors. The expressed and purified subunit after its engineered modification, may be useful for preventing hypertension and for other medical purposes.
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Inibidores da Enzima Conversora de Angiotensina/metabolismo , Antígenos de Plantas/metabolismo , Escherichia coli/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Antígenos de Plantas/genética , Antígenos de Plantas/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Peptidil Dipeptidase A/metabolismo , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/isolamento & purificaçãoRESUMO
En este trabajo se analiza la influencia que ejerce el mercado en la actividad físico-deportiva, misma que en la actualidad aparece asociada con una mayor atención sobre el cuerpo. Evidencias documentales y empíricas muestran que a través de la actividad físico-deportiva, el mercado impone, entre otros aspectos, las ventajas de la apariencia legítima dirigidas a fortalecer las relaciones interpersonales y la supremacía del atractivo físico como parámetro de aceptabilidad social y como medida del valor de cambio entre los individuos.
In this paper we analyzed the influence of the market in physical activity and sport, which currently appears associated with greater attention to the human body. Document analysis and empirical evidence show that through physical activity and sport, the market asserts the advantages of an ideal/legitimate appearance for strengthening interpersonal relationships and for advancing the supremacy of physical attractiveness both as a parameter of social acceptability and a measure of value of exchange between individuals.
Este trabalho analisa a influência que exerce o mercado na atividade física esportiva, mesmo que na atualidade apareça associada como uma atenção destinada ao corpo. Evidências documentadas e empíricas mostram que através da atividade física esportiva o mercado impõe entre outros aspectos, vantagens da aparência legítima dirigida ao fortalecimento das relações interpessoais e supremacia do atrativo físico, como parâmetro de aceitação social e como medida de valor de transformação entre os indivíduos.
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Humanos , Imagem Corporal , Técnicas de Exercício e de Movimento , Sociologia , EsportesRESUMO
Amarantin acidic subunit has the potential to be employed as a functional and a nutraceutical protein. To evaluate both possibilities this protein was produced in recombinant Escherichia coli Origami (DE3) harboring the expression plasmid pET-AC6His. Three different expression factors were assayed: inductor concentration, temperature and time of the amarantin acidic subunit accumulation. The results indicated that a 0.3 mmol/L concentration of isopropyl-beta-D-thiogalactoside, at 37 degrees C and 6 h after induction were favorable for high expression of amarantin acidic subunit, mostly in the form of inclusion bodies. The protein was purified from soluble fraction by immobilized metal affinity chromatography, up to 30 mg amarantin acidic subunit/L Terrific broth culture were obtained. Sucrose density gradient ultracentrifugation analysis of the expressed soluble amarantin acidic subunit revealed that it was assembled in monomers. The expression of the amarantin acidic subunit, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.
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Amaranthus/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Amaranthus/genética , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Espectrometria de Massas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sementes/genéticaRESUMO
The methylotrophic yeast Pichia pastoris was used to express an 11S seed globulin from Amaranthus hypochondriacus. Three different plasmids were tested for expression of amarantin. One of them, which included the untranslated regions (UTR) of the full cDNA, failed to express the amarantin under tested conditions, whereas the other plasmids, one without UTR and the other similar but including the endoplasmic reticulum-retention signal KDEL, were able to express the proamarantin in P. pastoris. After 48 h of induction, KDEL-proamarantin had accumulated quite significantly compared to unmodified proamarantin. Different solubilization patterns were also obtained from both proamarantin versions; only soluble protein was obtained from the system that included the KDEL retrieval signal. Protein fractionation was carried out by differential precipitation with ammonium sulfate, and proamarantin purification was performed using an HPLC ion exchange column. The endoplasmic reticulum-retention C-terminal sequence (KDEL retrieval signal), not commonly employed in this heterologous expression system, can therefore be used to enhance accumulation of recalcitrant protein in P. pastoris. The results obtained here also suggest that this expression system is suitable for expression and evaluation of engineered seed globulin proteins.