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Identification, Sequencing, Expression and Evolutionary Relationships of the 11S Seed Storage Protein Gene in Chenopodium quinoa Willd.Balzotti, Marie Renee Barrett 20 July 2006 (has links) (PDF)
Quinoa (Chenopodium quinoa) is an Andean crop adapted to harsh environmental conditions and containing a high and well-balanced protein composition. Two seed storage proteins, the 2S albumin and 11S globulin, are the major amino acid reservoirs for the developing seedling. An in-depth study of the genes encoding these proteins is necessary to understand the source of quinoa's protein quality. Our objectives include identification and sequencing of the 11S genomic and cDNA clones, analysis of 11S expression profiles in different quinoa accessions and evaluation of evolutionary relationships between the sequence of the 11S gene in quinoa and homologous genes in other species. Clones containing 11S cDNA and genomic DNA were identified and sequenced. Two copies of the gene were found to be present at two different loci in the quinoa genome. The amino acid composition of the 11S gene was also analyzed. Results show that the 11S gene contains a well-balanced assortment of essential amino acids with relatively high levels of glutamate/glutamine, aspartate/asparagine, arginine, serine, leucine and glycine, typical of other 11S seed storage proteins. Total RNA and globulin was extracted from seed collected at different developmental intervals from nine quinoa accessions. Expression profiles were determined by evaluating 11S transcript levels using relative quantification real time RT-PCR and comparing relative 11S globulin accumulation using sodium SDS-PAGE. The 11S gene was found to be expressed during late-maturation regardless of differences in maturation rate. A portion of the amino acid sequence of the 11S and homologous genes was chosen for phylogenetic analysis. Fifty five such sequences from 50 different plant species were assembled and aligned. Two phylogenetic reconstructions, one using the parsimony method and the other using Bayesian analysis, were generated in order to analyze evolutionary relationships between the 11S gene in quinoa and homologous genes in other species. Relationships shown by both reconstructions for sequences from closely-related species were consistent with taxonomic clustering. Both reconstructions showed less resolution involving relationships between distantly-related angiosperm taxa indicating a wide divergence of sequence at the angiosperm level and a need for additional angiosperm sequence data for finer resolution.
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Determinação dos componentes alergênicos da proteina isolada da soja / Determination of allergenic components isolated soy proteinBittencourt, Alvorita Leite 25 March 2002 (has links)
O objetivo deste trabalho foi esclarecer qual das frações protéicas da soja é mais alergênica, avaliando-se sua imunogenicidade e alergenicidade por imunoensaios e teste de anafilaxia cutânea passiva. Além desse propósito, este estudo visou a produção de anticorpos monoclonais para utilizá-lo como ferramenta na padronização de um ensaio imunoenzimático com a finalidade de detectar proteínas da soja, em produtos comercializados. A purificação das frações 2S,7S e 11S da soja foi realizada com base em estudos prévios, padronizando-se a metodologia dentro das condições experimentais do laboratório. A constatação da pureza foi realizada por eletroforese em gel de poliacrilamida (gel de empilhamento a 5% e de separação em gradiente de 7 a 15%), onde se verificou as bandas protéicas características de cada fração. Evidenciou-se as subunidades α (63,17 KDa) α\' (58,06 KDa) e β (42,09 KDa), correspondentes à fração 7S da soja e as subunidades ácida (38,8 KDa) e básica (21,04 KDa), correspondentes à fração 11S da soja. A fração 2S da soja mostrou uma banda protéica de 20 KDa, nesse ensaio. Os resultados da cinética demonstraram que as frações 7S e 11S da soja são muito imunogênicas, visto que induziram uma grande produção de anticorpos das classes IgM e IgG, em camundongos BALB/c. Por outro lado, a fração 2S da soja induziu uma pequena produção de anticorpos IgG, principalmente no 30º dia após a imunização. No teste de anafilaxia cutânea passiva, quando se avaliou a capacidade das frações protéicas da soja de induzirem a produção de anticorpos IgE, em camundongos BALB/c, verificou-se que a fração 7S da soja é alergênica, nesse modelo experimental. Entretanto, as frações 2S e 11S não estimularam o sistema imunológico desses animais a produzir essa classe de anticorpo. Quando se investigou a presença de anticorpos IgE dirigidos contra as frações protéicas 2S, 7S e 11S da soja, no soro de pacientes alérgicos, obteve-se resultado negativo com todas as três proteínas estudadas. Entretanto, na análise da presença de IgG, tanto o soro dos pacientes alérgicos como o dos controles mostraram-se reativos às frações 2S e 7S da soja, sugerindo a presença do anticorpo IgG4 anafilático no soro teste. Os quatro anticorpos monoclonais reativos às frações 7S e 11S da soja, obtidos neste estudo, mostraram-se reativos até as diluições 1/8000 (2A8 e 1H4) e 1/10000 (1F9) para a fração 7S e 1/12000 (3F2) para a fração 11S da soja. A identificação dos determinantes antigênicos reconhecidos pelos anticorpos monoclonais foi realizado por Imunotransferência. Observou-se que os anticorpos monoclonais anti-fração protéica 7S da soja (1H4, 2A8 e 1F9) reconheceram as subunidades α, α e β (1F9), α\' e β (1H4) e β (2A8), além de outras proteínas. Por outro lado, o anticorpo monoclonal anti-fração protéica 11S da soja (3F2) reagiu apenas com a subunidade básica dessa proteína. A padronização do ensaio imunoenzimático com o objetivo de detectar proteínas de soja, em produtos comercializados, mostrou que os anticorpos monoclonais dirigidos contra as frações protéicas 7S e 11S reconhecem, apenas, as proteínas da soja. Em conclusão, esses resultados sugerem que as frações protéicas 7S e 11S da soja são imunogênicas em camundongos, enquanto que na espécie humana, essa resposta foi observada com as frações 2S e 7S da soja. No teste de anafilaxia cutânea passiva, a fração protéica 7S da soja mostrou a capacidade de induzir anticorpos da classe IgE em camundongos BALB/c, sugerindo que essa fração é alergênica nesse ensaio. Por outro lado, os anticorpos monoclonais reativos às frações 7S e 11S detectaram as proteínas da soja nos produtos comercializados testados. / The goal of this study was to elucidate which of the soy protein fractions is more allergenic by using enzyme immunoassays and passive cutaneous anaphylactic activity test. Furthermore, monoclonal antibodies were obtained in order to standardize immunoassays to detect soy protein fractions in commercial soy-derived products. Purification of 2S, 7S and 11S fractions from soy was based on previous studies which were adapted to our experimental conditions. Purity of the isolated fractions was determined by polyacrilamide gel electrophoresis (7-15%). The subunits α (63,17 KDa) α\' (58,06 KDa) and β (42,09 KDa), corresponding to 7S fraction as well as the acid (38,8 KDa) and basic (21,04 Kda) subunits of 11S fraction were visualized. The 2S fraction showed a 20 KDa band only. Data showed that the 7S and 11S soy fractions are immunogenic as they elicited IgM and IgG antibodies in BALB/c mice. In contrast, the 2S fraction was not immunogenic in this specie. The passive cutaneous anaphylactic activity test showed that 7S fraction is allergenic as it elicited IgE production in BALC mice. However, this test was negative for 2S and 7S fractions. No IgE reactive against the 2S, 7S e 11S was found in sera of patients (n=5) presenting previous food allergy symptoms. However, either these patients or controls (n=5) showed IgG reactive against 2S and 7S fractions in their sera. The four monoclonal antibodies obtained in this study were reactive up to 1/8000 (2A8 and 1H4) and 1/10000 (1F9) against 7S fraction, and up to 1/12000 (3F2) for 11S fraction. Immunoblot analysis showed that the monoclonal antibodies anti-7S fraction (1H4, 2A8 e 1F9) recognized the α α\' and β (1F9), α\' and β (1H4) and α (2A8) subunits. The monoclonal antibody anti-11S fraction (3F2) reacted with the basic subunit of this fraction only. By using ELISA and the monoclonal antibodies anti-7S and anti-11S it was possible to detect especifically these soy protein fractions in commercial soy-derived products. In conclusion, data suggest that 7S and 11S soy protein fractions are immunogenic in mice while 2S and 7S fraction are immunogenic in humans. The IgE response to 7S fraction in BALB/c mice showed by the passive cutaneous anaphylactic activity test indicates that this soy protein fraction is allergenic in this experimental model. Finally, the monoclonal antibodies anti-7S and anti-11S obtained in this study did not react with other vegetable or animal proteins being suitable to be used in ELISA for detection of these proteins in commercial soy-derived products.
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Determinação dos componentes alergênicos da proteina isolada da soja / Determination of allergenic components isolated soy proteinAlvorita Leite Bittencourt 25 March 2002 (has links)
O objetivo deste trabalho foi esclarecer qual das frações protéicas da soja é mais alergênica, avaliando-se sua imunogenicidade e alergenicidade por imunoensaios e teste de anafilaxia cutânea passiva. Além desse propósito, este estudo visou a produção de anticorpos monoclonais para utilizá-lo como ferramenta na padronização de um ensaio imunoenzimático com a finalidade de detectar proteínas da soja, em produtos comercializados. A purificação das frações 2S,7S e 11S da soja foi realizada com base em estudos prévios, padronizando-se a metodologia dentro das condições experimentais do laboratório. A constatação da pureza foi realizada por eletroforese em gel de poliacrilamida (gel de empilhamento a 5% e de separação em gradiente de 7 a 15%), onde se verificou as bandas protéicas características de cada fração. Evidenciou-se as subunidades α (63,17 KDa) α\' (58,06 KDa) e β (42,09 KDa), correspondentes à fração 7S da soja e as subunidades ácida (38,8 KDa) e básica (21,04 KDa), correspondentes à fração 11S da soja. A fração 2S da soja mostrou uma banda protéica de 20 KDa, nesse ensaio. Os resultados da cinética demonstraram que as frações 7S e 11S da soja são muito imunogênicas, visto que induziram uma grande produção de anticorpos das classes IgM e IgG, em camundongos BALB/c. Por outro lado, a fração 2S da soja induziu uma pequena produção de anticorpos IgG, principalmente no 30º dia após a imunização. No teste de anafilaxia cutânea passiva, quando se avaliou a capacidade das frações protéicas da soja de induzirem a produção de anticorpos IgE, em camundongos BALB/c, verificou-se que a fração 7S da soja é alergênica, nesse modelo experimental. Entretanto, as frações 2S e 11S não estimularam o sistema imunológico desses animais a produzir essa classe de anticorpo. Quando se investigou a presença de anticorpos IgE dirigidos contra as frações protéicas 2S, 7S e 11S da soja, no soro de pacientes alérgicos, obteve-se resultado negativo com todas as três proteínas estudadas. Entretanto, na análise da presença de IgG, tanto o soro dos pacientes alérgicos como o dos controles mostraram-se reativos às frações 2S e 7S da soja, sugerindo a presença do anticorpo IgG4 anafilático no soro teste. Os quatro anticorpos monoclonais reativos às frações 7S e 11S da soja, obtidos neste estudo, mostraram-se reativos até as diluições 1/8000 (2A8 e 1H4) e 1/10000 (1F9) para a fração 7S e 1/12000 (3F2) para a fração 11S da soja. A identificação dos determinantes antigênicos reconhecidos pelos anticorpos monoclonais foi realizado por Imunotransferência. Observou-se que os anticorpos monoclonais anti-fração protéica 7S da soja (1H4, 2A8 e 1F9) reconheceram as subunidades α, α e β (1F9), α\' e β (1H4) e β (2A8), além de outras proteínas. Por outro lado, o anticorpo monoclonal anti-fração protéica 11S da soja (3F2) reagiu apenas com a subunidade básica dessa proteína. A padronização do ensaio imunoenzimático com o objetivo de detectar proteínas de soja, em produtos comercializados, mostrou que os anticorpos monoclonais dirigidos contra as frações protéicas 7S e 11S reconhecem, apenas, as proteínas da soja. Em conclusão, esses resultados sugerem que as frações protéicas 7S e 11S da soja são imunogênicas em camundongos, enquanto que na espécie humana, essa resposta foi observada com as frações 2S e 7S da soja. No teste de anafilaxia cutânea passiva, a fração protéica 7S da soja mostrou a capacidade de induzir anticorpos da classe IgE em camundongos BALB/c, sugerindo que essa fração é alergênica nesse ensaio. Por outro lado, os anticorpos monoclonais reativos às frações 7S e 11S detectaram as proteínas da soja nos produtos comercializados testados. / The goal of this study was to elucidate which of the soy protein fractions is more allergenic by using enzyme immunoassays and passive cutaneous anaphylactic activity test. Furthermore, monoclonal antibodies were obtained in order to standardize immunoassays to detect soy protein fractions in commercial soy-derived products. Purification of 2S, 7S and 11S fractions from soy was based on previous studies which were adapted to our experimental conditions. Purity of the isolated fractions was determined by polyacrilamide gel electrophoresis (7-15%). The subunits α (63,17 KDa) α\' (58,06 KDa) and β (42,09 KDa), corresponding to 7S fraction as well as the acid (38,8 KDa) and basic (21,04 Kda) subunits of 11S fraction were visualized. The 2S fraction showed a 20 KDa band only. Data showed that the 7S and 11S soy fractions are immunogenic as they elicited IgM and IgG antibodies in BALB/c mice. In contrast, the 2S fraction was not immunogenic in this specie. The passive cutaneous anaphylactic activity test showed that 7S fraction is allergenic as it elicited IgE production in BALC mice. However, this test was negative for 2S and 7S fractions. No IgE reactive against the 2S, 7S e 11S was found in sera of patients (n=5) presenting previous food allergy symptoms. However, either these patients or controls (n=5) showed IgG reactive against 2S and 7S fractions in their sera. The four monoclonal antibodies obtained in this study were reactive up to 1/8000 (2A8 and 1H4) and 1/10000 (1F9) against 7S fraction, and up to 1/12000 (3F2) for 11S fraction. Immunoblot analysis showed that the monoclonal antibodies anti-7S fraction (1H4, 2A8 e 1F9) recognized the α α\' and β (1F9), α\' and β (1H4) and α (2A8) subunits. The monoclonal antibody anti-11S fraction (3F2) reacted with the basic subunit of this fraction only. By using ELISA and the monoclonal antibodies anti-7S and anti-11S it was possible to detect especifically these soy protein fractions in commercial soy-derived products. In conclusion, data suggest that 7S and 11S soy protein fractions are immunogenic in mice while 2S and 7S fraction are immunogenic in humans. The IgE response to 7S fraction in BALB/c mice showed by the passive cutaneous anaphylactic activity test indicates that this soy protein fraction is allergenic in this experimental model. Finally, the monoclonal antibodies anti-7S and anti-11S obtained in this study did not react with other vegetable or animal proteins being suitable to be used in ELISA for detection of these proteins in commercial soy-derived products.
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Quality Testing and Selection of Soybeans for Cultivation in Mississippi for Soymilk and Tofu ProductionMeng, Shi 14 August 2015 (has links)
Soybeans with large seed size, uniformity, clear hilum, and high 11S/7S ratio are favored for soymilk and tofu making. In order to find ideal soybean lines for food making, sixty-eight soybean lines, which were selected from thousands of accessions in the USDA-Soybean Germplasm Collection, were planted in three successive seasons. Eight lines were identified from twenty-two lines harvested in 2014 (Stoneville, MS) to be suitable for tofu making as judged by chemical composition and sensory quality of tofu. The results provided important food quality information to the growers, breeders and tofu industries for their selection of soybean to improve food quality. In the filled tofu making and texture analysis study, the correlation between A3 subunit percentage and tofu firmness was significant (N=22. r = 0.77, P < 0.001). The result proved that the percentage of A3 subunits could be an indicator for predicting the firmness of tofu.
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Der Einfluss des HIV-1 Tat-Proteins auf das Proteasom-System und die Folgen für die zelluläre ImmunabwehrHuang, Xiaohua 06 June 2002 (has links)
Das HIV-1 Tat-Protein hemmt die Peptidase-Aktivität des 20S Proteasoms durch Konkurrenz mit dem 11S Regulator/PA28 um die Bindungsstelle am Proteasom. Aus den kinetischen Daten und durch Strukturvergleiche geht hervor, dass die Aminosäuren Lys51, Arg52 und Asp67 des Tat-Proteins für den Effekt auf das 20S Proteasom verantwortlich sind und die REG/Tat-Proteasom-Bindungsstelle bilden. Eine in der 11S Regulator alpha-Untereinheit (REG alpha) identifizierte vergleichbare Struktur wird von den Aminosäuren Glu235, Lys236 und Lys239 gebildet. Durch eine Mutation der REG alpha Aminosäuren Glu235 und Lys236 zu Ala geht die Fähigkeit des REG alpha die Peptidase-Aktivität des 20S Proteasoms zu stimulieren verloren, während die Bindungsfähigkeit an den 20S Komplex erhalten bleibt. Die Bindungsstelle in REG alpha ist für die verstärkte Präsentation eines Epitops des Cytomegalovirus pp89 durch MHC Klasse I essentiell. Das Tat-Protein und das Tat-Peptid 37-72 unterdrücken die 11S-Regulator vermittelte Antigenpräsentation des pp89 Epitops. Im Gegensatz dazu weist das Tat-Peptid mit Mutation der Aminosäuren Lys51, Arg52 und Asp67 zu Ala keine Reduktion der Antigenpräsentation auf. / The HIV-1 Tat protein inhibits the peptidase activity of the 20S proteasome and competes with the 11S regulator/PA28. Kinetic assays and structural comparison found amino acids Lys51, Arg52 and Asp67 of Tat to be responsible for the effects on proteasomes, forming the REG/Tat-proteasome-binding site. A similar site identified in the 11S regulator alpha subunit (REG alpha) consists of the residues Glu235, Lys236 and Lys239. Mutation of the REG alpha amino acids Glu235 and Lys236 to Ala resulted in a REG alpha mutant that lost the ability to activate the 20S proteasome even though it still binds to the 20S complex. The site in REG alpha is needed to enhance the presentation of a cytomegalovirus pp89 protein-derived epitope by MHC class I molecules. Full-length Tat and the Tat peptide 37-72 suppressed 11S regulator-mediated presentation of the pp89 epitope. In contrast, a Tat peptide with mutation of amino acids Lys51, Arg52 and Asp67 to Ala was not able to reduce antigen presentation.
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STRUCTURE AND PROPERTIES OF CRUCIFERIN: INVESTIGATION OF HOMOHEXAMERIC CRUCIFERIN EXPRESSED IN ARABIDOPSIS2013 June 1900 (has links)
The structure of 11S cruciferin has been solved; however, how the individual subunits contribute to its physico-chemical and functional properties are not well known. The cruciferin isoforms in Arabidopsis thaliana, CRUA, CRUB, and CRUC, were investigated with respect to their molecular structures and the relationship of structural features to the physico-chemical and functional properties of cruciferin using homology modeling and various analytical techniques.
Comparison of these models revealed that hydrophobicity and electrostatic potential distribution on the surface of the CRUC homotrimer had more favorable interfacial, solubility, and thermal properties than those of CRUA or CRUB. Flavor binding and pepsin digestion were associated with hypervariable regions (HVRs) and center core regions, respectively, moreso for CRUA and CRUB homotrimers than for CRUC.
Chemical imaging of a single cell area in wild type (WT) and double-knockout seeds (CRUAbc, CRUaBc, and CRUabC) using synchrotron FT-IR microscopy (amide I band, 1650 cm-1, νC=O) showed that seed storage proteins were concentrated in the cell center and protein storage vacuoles, whereas lipids were closer to the cell wall. Secondary structure components of proteins of double-knockout lines did not show major differences. Changes in protein secondary structure components of pepsin-treated CRUabC (CRUC) mutant were minimal, indicating low enzyme accessibility.
A three-step chromatographic procedure allowed isolation of the hexameric form of cruciferin with high purity (>95%). Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic analysis of the secondary structure of these proteins revealed cruciferins were folded into higher order secondary structures; 44−50% β-sheets and 7−9% α-helices. The relative subunit ratio was approximately 1:3:6 (CRUA:CRUB:CRUC) in the WT cruciferin. The Tm values of purified cruciferin at pH 7.4 (μ = 0.0) were in the order of WT = CRUA = CRUB < CRUC. The order of surface hydrophobicity as determined by ANS (1-anilinonaphthalene-8-sulfonate) probe binding was CRUA > CRUB = WT >> CRUC.
Intrinsic fluorescence studies revealed a compact molecular structure for the CRUC homohexamer compared to the CRUA and CRUB homohexamers. The order of emulsion forming abilities was CRUA = CRUB > WT > CRUC (no emulsion formation) and the order of heat-induced network structure strength was WT > CRUA = CRUB > CRUC (no gel formation). The inability of CRUC to form gels or emulsions may be attributed to its low surface hydrophobicity and molecular compactness. At pH 2.0, CRUC hexamers dissociated into trimers which allowed the formation of an O/W emulsion and heat-induced network structures.
Solubility of cruciferin as a function of pH at low ionic strength gave two minima around pH 4 and 7.4 yielding a “W” shape solubility profile deviating from the typical “U” or “V” shape solubility profile of other 11S globulins. The high ionic strength (μ = 0.5) was not favorable for emulsification, heat-induced gel formation, or solubilization for all cruciferins. Furthermore, the CRUA and CRUB homohexamers exhibited rapid pepsinolysis, while the CRUC homohexamer and WT heterohexamer were digested more slowly.
Although fairly well conserved regions were found in the primary structure of these three cruciferin subunits, differences were found in the hypervariable regions and extended loop regions resulting in slight differences in 3D structures and interactions that occur during association to form superstructures, such as hexamers. These differences were reflected in the physico-chemical and techno-functional properties of hexamers and trimers composed of each subunit. In silico predictions for certain functionalities were highly correlated with empirical data from laboratory experiments.
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