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Soluble and bound peroxidases from papaya fruit

da Silva, Elisete; Lourenco, Euclides J.; Neves, Valdir A.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 1051-1056
ENG
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Soluble and bound peroxidases were isolated from the pulp of ripening papaya fruit. During papaya ripening, soluble and bound peroxidase activities increased 2.5- and 4.2-fold, respectively. Soluble peroxidase was purified 59-fold by ammonium sulphate precipitation and chromatography on Sephadex G-25, DEAE-cellulose and Sephadex G-100. Bound peroxidase was purified 140-fold by ammonium sulphate precipitation and chromatography on Sephadex G-100 and DEAE-cellulose. Polyacrylamide gel electrophoresis of the purified preparations revealed that both enzymes were highly purified by the procedures adopted. The soluble and bound forms had a Mr of 41 000 and 54 000, respectively. Soluble and bound peroxidases showed optimum activity at pH 6.0 and 5.5, respectively, and were inhibited by p-chloromercuribenzoate, iodoacetamide, N-ethylmaleimide, potassium cyanide and Fe2+. Soluble peroxidase was activated by ammonium sulphate and this activation was prevented by cyanide. © 1990.

O isotipo IgG de artiodactilos : as imunoglobinas G do Pecari brasileiro, Tayassu tajacu (Linne, 1758)

Maria de Fatima Lovo Farah
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em //1988 PT
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A análise imunoeletroforética do soro imune de Tayassu, utilizando-se como revelador o soro de coelho anti-soro de Tayassu, revelou uma fração protéica de mobilidade eletroforética menos anódina característica de imunoglobulinas do isotipo IgG. Do soro imune deste animal foi purificada, por cromatografia de troca iônica em DEAE celulose, uma fração protéica que por análise imunoeletroforética apresentou mobilidade eletroforética lenta em relação ao anodo. Da fração restante, denominada RC, foi purificada a fração protéica, por cromatografia de exclusão molecular, que apresentou volume de eluição típico de imunoglobulinas IgG. A análise imunoeletroforética demonstrou que a fração obtida por purificação do RC, possui mobilidade eletroforética rápida em relação ao anodo. Por imunodifusão, as frações protéicas denominadas IgG lenta e IgG rápida de Tayassu apresentaram relação de identidade parcial com a IgG monoclonal GOB quando reveladas com o soro especifico anticadeia gama humana, evidenciando que estas proteínas são parte integrante da classe IgG de vertebrados. A digestão pela papaína, da IgG lenta de Tayassu, permitiu a separação desta imunoglobulina nos fragmentos Fab e Fc, por cromatografia de troca iônica em DEAE celulose. Estes resultados indicam que as imunoglobulinas Gde Tayassu devem guardar relação de semelhança estrutural com as demais imunoglobulinas de vertebrados. Por imunodifusão...

Estudo da fração fosfolipasica A2 isolada do veneno de Bothrops insularis na junção neuromuscular

Jose Carlos Cogo
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 10/04/1995 PT
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Duas frações com atividade fosfolipásica (?PLA IND. 2?) foram isoladas do veneno total de Bothrops insularis através de 2 processos de fracionamento: gel de Sephadex G-75 em tampão formato de amônio 50 mM, pH 3,5 e gel de Sephadex G-150 em tampão fosfato salina (PBS) 0,1M, pH 7,2 seguido por cromatografia de troca iônica em DEAE Sephadex em tampão bicarbonato de amônio 0,01 M, pH 8,0. A fração isolada em pH ácido mostrou-se menos efetiva que a fração obtida em pH neutro. Esta última reproduziu todos os efeitos farmacológicos observados com o veneno total. Estes foram associados apenas à atividade (?PLA IND. 2?) pois a fração é desprovida de atividades esterásica, coagulante e caseinolítica. Utilizando técnicas de eletrofisiologia, miografia, determinação dos níveis de creatino quinase (CQ) e estudos das alterações morfológicas (músculos incubados com r- a fração (?PLA IND. 2?) ou injetados com o veneno e fração (?PLA IND. 2?) e comparando nossos resultados com aqueles existentes na literatura, chegamos às seguintes conclusões: 1) Alterações miográficas induzidas pela fração (?PLA IND. 2?) isolada em pH neutro,caracterizam-se pela facilitação da neurotransmissão (aumento da amplitude de contração muscular e aumento da freqüência dos potenciais em miniatura da placa terminal) seguida por bloqueio das contrações musculares. O bloqueio induzido por esta fração...

Some toxinological aspects of Aurelia aurita (Linné) from the Mexican Caribbean

Segura-Puertas,L.; Avila-Soria,G.; Sánchez-Rodríguez,J.; Ramos-Aguilar,M. E.; Burnett,J. W.
Fonte: Centro de Estudos de Venenos e Animais Peçonhentos - CEVAP, Universidade Estadual Paulista - UNESP Publicador: Centro de Estudos de Venenos e Animais Peçonhentos - CEVAP, Universidade Estadual Paulista - UNESP
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2002 EN
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Aurelia aurita is a scyphozoan, abundant in the Mexican Caribbean during summer. Although usually innocuous, there is evidence of it causing harm to humans. This work investigates the biological activities of crude and fractionated extracts of A. aurita. Live specimens were collected between July and September 1999 from the Mexican Caribbean. The tentacular margin was dissected immediately and frozen at -50ºC. A nematocyst suspension was prepared, discharged, and the supernatants lyophilized. Hemolytic assay was performed with lyophilized crude extract on bovine, sheep, and human red blood cells. Erythrocyte sensitivity to the toxin was ranked in descending order: human, sheep, and bovine. Toxic activity on Artemia nauplii was evaluated using the same crude extract for different exposure periods (3, 5, and 10 hours); only 48 and 72 hour old Artemia nauplii showed 50% mortality. Partial toxin purification was completed by sequential liquid chromatography using three gels (Sephadex G-200, DEAE Sephadex A-50, and Sephadex G-100). Intramuscular neuroactivity was detected in the crab Ocypode quadrata for two partially purified fractions. These fractions were found to have molecular weight components of 66 and 45 kDa, respectively.

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

Miller, D. N.; Bryant, J. E.; Madsen, E. L.; Ghiorse, W. C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/1999 EN
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We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding...

Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

Bannerman, E N; Nicolet, J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1976 EN
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The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase.

Purification and Properties of β-1, 4-Xylanase from Aeromonas caviae W-61

Viet, Dung Nguyen; Kamio, Yoshiyuki; Abe, Naoki; Kaneko, Jun; Izaki, Kazuo
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1991 EN
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Aeromonas caviae W-61, which was isolated from water samples at the Faculty of Agriculture, Tohoku University, produced β-1, 4-xylanase (1,4-β-d-xylan xylanohydrolase; EC 3.2.1.8) extracellularly. The xylanase was purified to homogeneity by using DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-100 column chromatographies. The molecular weight of the purified enzyme was estimated to be 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 9.2. The optimal pH and temperature for the activity of the enzyme were 7.0 and 55°C, respectively. The enzyme was stable at pH 7.0 at temperatures of up to 50°C. As enzymatic products, various xylo-oligosaccharides such as xylobiose, xylotriose, xylotetraose, and xylopentaose were formed, and only a small amount of xylose was detected. The purified enzyme did not hydrolyze starch, cellulose, carboxymethylcellulose, or β-1, 3-xylan.

Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into the di-chain form.

Dekleva, M L; Dasgupta, B R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1990 EN
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A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.

Separation and distribution of thiosulfate-oxidizing enzyme, tetrathionate reductase, and thiosulfate reductase in extracts of marine heterotroph strain 16B.

Whited, G M; Tuttle, J H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1983 EN
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Thiosulfate-oxidizing enzyme (TSO), tetrathionate reductase (TTR), and thiosulfate reductase (TSR) were demonstrated in cell-free extracts of the marine heterotrophic thiosulfate-oxidizing bacterium strain 16B. Extracts prepared from cells cultured aerobically in the absence of thiosulfate or tetrathionate exhibited constitutive TSO and TTR activity which resided in the soluble fraction of ultracentrifuged crude extracts. Constitutive TSO and TTR cochromatographed on DEAE-Sephadex A-50, Cellex D, Sephadex G-150, and orange A dye-ligand affinity gels. Extracts prepared from cells cultured anaerobically with tetrathionate or aerobically with thiosulfate followed by oxygen deprivation showed an 11- to 30-fold increase in TTR activity, with no increase in TSO activity. The inducible TTR resided in both the ultracentrifuge pellet and supernatant fractions and was readily separated from constitutive TSO and TTR in the latter by DEAE-Sephadex chromatography. Inducible TTR exhibited TSR activity, which was also located in both membrane and soluble extract fractions and which cochromatographed with inducible TTR. The results indicate that constitutive TSO and TTR in marine heterotroph 16B represent reverse activities of the same enzyme whose major physiological function is thiosulfate oxidation. Evidence is also presented which suggests a possible association of inducible TTR and TSR in strain 16B.

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Friedrich, B; Friedrich, C G; Schlegel, H G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1976 EN
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Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate...

Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum

De Mot, René; Verachtert, Hubert
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1985 EN
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The extracellular amylolytic system of Filobasidium capsuligenum consisted of an α-amylase (1,4-α-d-glucan glucanhydrolase, EC 3.2.1.1) and two forms of glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3). The enzymes were purified by ammonium sulfate fractionation, repeated ion-exchange chromatography (DEAE-Sephadex A-50), and gel filtration (Sephadex G-25, Sephadex G-100 sf). α-Amylase had an optimum pH of 5.6 and an optimum temperature of 50°C but was rapidly inactivated at higher temperature. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 64,000. An acarbose concentration of 20 μg/ml was required for 50% inhibition of the α-amylase. Both glucoamylases are glycoproteins of identical molecular weight (60,000) and produce only glucose by exohydrolysis. The debranching activity of the glucoamylases was evidenced with substrates containing α-1,6 linkages. The pH optima were 5.0 to 5.6 for glucoamylase I and 4.8 to 5.3 for glucoamylase II. Glucoamylase I had a higher optimum temperature (55°C) than glucoamylase II (50°C) and was also more resistant to thermal inactivation. Only low acarbose concentrations (<0.1 μg/ml) were required to reduce the activity of the glucoamylases by 50%.

Produção e caracterização parcial de um composto de baixa massa molecular com atividade fenoloxidasica, de Thermoascus aurantiacus

Angela Elena Machuca Herrera
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 16/10/1995 PT
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No presente trabalho estudou-se a produção de extratos com atividade fenoloxidásica (FOx) pelo fungo termófilo lhermoasclls aurantiacus. Foram determinadas as melhores condições de cultivo para produção da atividade FOx em meio líquido. A adição de substratos polissacarídicos induziu altos teores de atividade, quando comparado com cultura contendo somente glicose. Farelo de trigo foi o melhor substrato, e em concentração de 1,5% (m/v) induziu níveis entre 1 a 2 UI/mL de atividade. O tipo e tamanho do inóculo afetaram significativamente a produção de atividade, porém a agitação e a oxigenação das culturas não tiveram efeitos significativos. Um pH inicial do meio de cultura entre 6 e 8 favoreceu a produção de atividade FOx. Quando substratos polissacarídicos foram adicionados à cultura, diversas atividades enzimáticas relacionadas à degradação de lignina, em menor ou maior grau, foram detectadas: lacase, peroxidase, manganês-peroxidase, celobiose-quinona oxidoreductase e álcool veratrílico-oxidase. Nas mesmas condições não foi detectada atividade lignina-peroxidase. Após estabelecer as melhores condições para produção de atividade FOx pelo Taurantiacus, foram produzidos extratos com alta atividade para estudos de caracterização cinética. A atividade FOx presente no extrato bruto apresentou características semelhantes às fenoloxidases do tipo lacase. O extrato bruto oxidou uma variedade de substratos típicos de fenoloxidases...

Purificação e caracterização de uma proteina toxica isolada das glandulas submandibulares de camundongos machos

Maria Inez Fernandes Poletto
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 09/12/1997 PT
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Este trabalho foi realizado com o objetivo de isolar e carac terizar quimicamente uma proteína tóxica das glândulas submandibulares de camundongos machos, e observar histológica e macroscopicamente seu efeito biológico. Foram utilizadas 34 g de glândulas submandibulares de camundongos machos, de 3 meses de idade, para a obtenção do extrato glandular cru. Este extrato foi aplicado a uma coluna de DEAE Celulose equilibrada com tampão Tris HCI 0,05 M pH 7,6. A coluna foi lavada com 800 ml do mesmo tampão. O eluente obtido constitui a Fração I. Após a lavagem a coluna foi eluída seqüencialmente, com tampão Tris - NaCI 0,1 M (Fração, II) e tampão Tris - NaCI 0,3 M (Fração III). As frações protéicas foram concentradas por ultrafiltração e avaliada sua atividade tóxica e enzimática (Hidrólise do éster sintético BAPNA). A fração mais ativa (lI), foi dialisada contra tampão Tris - HCI pH 7,6 e aplicada a uma coluna de DEAE-Sephadex equilibrada com o mesmo tampão. As proteínas da coluna foram eluídas seqüencialmente por diferença de pH com os tampões Fosfato 0,1 M pH 7,0; 6,0 e 5,0. A fração mais ativa foi a obtida com tampão Fosfato 0,1 M pH 7,0 (`F IND. 2´ `P IND. 1´). Esta fração foi aplicada a uma coluna de Sephadex G-100 e as frações protéicas foram eluídas com o Tampão Tris HCI 0...

Propriedades fisico-quimicas e nutricionais de proteinas de feijão (Phaseolus vulgaris, L.) variedade Rosinha G2

Valdemiro Carlos Sgarbieri
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Livre Docência Formato: application/pdf
Publicado em //1979 PT
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O presente trabalho teve como principais objetivos o isolamento, o estudo da composição e do valor nutricional das principais frações protéicas do feijão (Phaseolus vulgaris, L) var. Rosinha G2, além da purificação e do estudo das propriedades dos inibidores da tripsina e da quimotripsina, presentes na fração albumina (F1). A extratibilidade das proteínas foi estudada em função do pH e da concentração salina ou força iônica do meio solvente. Em soluções ácidas (pH < 3) ou alcalinas (pH > 8) conseguiu-se extrair entre 85 e 9S% do nitrogênio total da farinha integral. A faixa de pH de menor extratibilidade foi entre 3,5 e 5,0. Duas extrações sucessivas da mesma amostra, a primeira com água destilada e a segunda com solução 0,5M de NaCl, extraia pelo menos 80% do nitrogênio da farinha integral. Extração fracionada da mesma farinha com vários solventes revelou que 481% do nitrogênio total era extraído com água destilada, 25,6% com solução 0,5M de NaCl, 19,3% com tampão borato de sódio pH 10,0, côn tendo 0,5% de mercaptoetanol e 0,5% de lauril sulfato de sódio, perfazendo a extração de 92,8% do nitrogênio total. Tratamento dos grãos a 97°C por vários intervalos de tempo mostrou que a solubilidade das proteínas em água diminuía de...

Estudo fitoquímico da espécie Serjania lethalis St. Hil.; Phytochemical study of the species Serjania lethalis St. Hil.

Pires, Edjane Vieira
Fonte: Universidade Federal de Alagoas; BR; Química; Biotecnologia; Programa de Pós-Graduação em Química e Biotecnologia; UFAL Publicador: Universidade Federal de Alagoas; BR; Química; Biotecnologia; Programa de Pós-Graduação em Química e Biotecnologia; UFAL
Tipo: Dissertação Formato: application/pdf
POR
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The brazilian is biodiversity considered one of higher of the world, is a bit know and explored as the source of drugs medicines. In search of new bioactive molecules the etanolic extract of stem of Serjania lethalis St. Hil was studied in for the molluscicide, miracicide and shistossomicida activity. The specie S. lethalis was collected in Brasilia and a voucher is deposited in the Herbarium of Federal University of Brasilia under the number JEP 3698 UB. After dried end reduced to powder the stem of S. lethalis was extracted in Soxhlet apparatus with ethanol 95%. The crude ethanol extract of stem was extracted, at low temperature, with acetone. The soluble fraction in acetona was purified through filtration in active charcol and permeation in sephadex gel LH 20 and additional preparative chromatographic plate for the obtention of saponin codified as SLS. An alternative method of separation involving the use of synthetic membranes that provide dialysis was used for hidrometanolic and ethyl acetate fractions obtained from the partition of the liquid-liquid crude ethanol extract (stem). Due to the antibacterial activity observed after dialysis follow up purification was made, using chromatography techniques on column with deacttived sílica and subsequent purification in Sephadex LH 20 column to obtain the SLD substance. The butanolic fraction obtained from liquid-liquid partition was active against Biomphalaria glabrata with CL90 10...

Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla

Smith, A. D.; Winkler, H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1967 EN
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1. A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25% of the total protein of the granules and gave a single band on gel electrophoresis. 2. The protein is unusually rich in acidic amino acids, notably glutamic acid (26·0%, w/w); it is also relatively rich in proline (8·6%, w/w) but poor in cystine (0·35%, w/w). 3. A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. 4. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. 5. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume. 6. The hydrodynamic properties of the protein...

IgE immune complexes stimulate arachidonic acid release by mouse peritoneal macrophages.

Rouzer, C A; Scott, W A; Hamill, A L; Liu, F T; Katz, D H; Cohn, Z A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1982 EN
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Resident mouse peritoneal macrophages were labeled with [3H]arachidonic acid and challenged with Sephadex beads coated with immune complexes of IgE and antigen. Arachidonic acid release by the cells was assessed by the quantity or radiolabel recovered from the culture medium. Freshly isolated macrophages responded to IgE immune complexes with a release of [3H]arachidonic acid that was linear for 1-2 hr. The magnitude of the response was dependent on both the number of immune complex-coated beads and on the degree of opsonization of the beads. Under conditions of maximal stimulation, macrophages challenged with IgE immune complex-coated Sephadex released 23 +/- 4.5% of their incorporated radiolabel. This is compared to values of 34.2 +/- 0.5% and 38.1 +/- 3.3% for cultures that received IgG immune complex-coated Sephadex or zymosan, respectively. Macrophages did not release arachidonic acid upon exposure to soluble IgE and antigen given sequentially or simultaneously, and soluble IgE did not inhibit the cells' response to IgE immune complexes. Incubation of macrophages for longer than 3 hr prior to challenge resulted in a selective loss in the cells' ability to respond to IgE immune complexes. After 16 hr of culture, macrophages released only 3.9 +/- 0.3% of their incorporated 3H on exposure to IgE immune complexes; however radiolabel release in response to zymosan (42.0 +/- 0.8%) was identical to that of freshly isolated cells. These data indicate that macrophages are capable of releasing arachidonic acid in response to preformed particulate immune complexes of IgE and antigen. Because Sephadex beads are too large to be interiorized by the cells...

Zinc binding in cow's milk and human milk.

Blakeborough, P; Salter, D N; Gurr, M I
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1983 EN
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In both cow's milk and human milk, zinc was associated with proteins of high molecular weight (greater than 100 000), as judged by analysis with Sephadex G-75. Precipitation of the casein at pH 4.6 and filtration of the resultant acid whey on Sephadex G-25 led, however, to the recovery of about 90% of the zinc as a compound of low molecular weight, which was tentatively identified as zinc citrate. Over 95% of the zinc of cow's milk was sedimented with the casein micelles on ultracentrifugation. Filtration of these micellar caseins on Sephadex G-150 gave two peaks containing zinc, which corresponded to aggregates of alpha-casein-kappa-casein and of alpha-casein-beta-casein. Ultracentrifugation of human milk sedimented only approx. 40% of total zinc. Analysis of sediment and supernatant on Sephadex G-150, however, indicated that about 85% of the zinc was associated with a protein complex of molecular weight greater than 150 000. The major protein of this complex was identified as lactoferrin. A minor zinc-binding component of average molecular weight 30 000 was also observed in the supernatant. The results indicated that zinc is bound to different macromolecules in cow's and human milk. This may be a factor affecting the bioavailability to the human infant of zinc from the two milks...

Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

Fleig, W E; Hoss, G; Nöther-Fleig, G; Ditschuneit, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1986 EN
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Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered...

Estudo fitoquímico da espécie Serjania lethalis St. Hil.; Phytochemical study of the species Serjania lethalis St. Hil.

Pires, Edjane Vieira
Fonte: Universidade Federal de Alagoas; BR; Química; Biotecnologia; Programa de Pós-Graduação em Química e Biotecnologia; UFAL Publicador: Universidade Federal de Alagoas; BR; Química; Biotecnologia; Programa de Pós-Graduação em Química e Biotecnologia; UFAL
Tipo: Dissertação Formato: application/pdf
POR
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The brazilian is biodiversity considered one of higher of the world, is a bit know and explored as the source of drugs medicines. In search of new bioactive molecules the etanolic extract of stem of Serjania lethalis St. Hil was studied in for the molluscicide, miracicide and shistossomicida activity. The specie S. lethalis was collected in Brasilia and a voucher is deposited in the Herbarium of Federal University of Brasilia under the number JEP 3698 UB. After dried end reduced to powder the stem of S. lethalis was extracted in Soxhlet apparatus with ethanol 95%. The crude ethanol extract of stem was extracted, at low temperature, with acetone. The soluble fraction in acetona was purified through filtration in active charcol and permeation in sephadex gel LH 20 and additional preparative chromatographic plate for the obtention of saponin codified as SLS. An alternative method of separation involving the use of synthetic membranes that provide dialysis was used for hidrometanolic and ethyl acetate fractions obtained from the partition of the liquid-liquid crude ethanol extract (stem). Due to the antibacterial activity observed after dialysis follow up purification was made, using chromatography techniques on column with deacttived sílica and subsequent purification in Sephadex LH 20 column to obtain the SLD substance. The butanolic fraction obtained from liquid-liquid partition was active against Biomphalaria glabrata with CL90 10...