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Localized, Positive Charge Mediates Adhesion of Rhodosporidium toruloides to Barley Leaves and Polystyrene

Buck, James W.; Andrews, John H.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1999 EN
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The physicochemical forces that mediate attachment of yeasts to the phylloplane are unknown. Cell surface charge and hydrophobicity and adhesion to polystyrene, glass, and barley were assessed for wild-type Rhodosporidium toruloides and attachment-minus (Att−) mutants. Cells were grown under conditions promoting (excess carbon) or not promoting (excess nitrogen) capsule production. Hydrophobicity was measured by adhesion to xylenes, and surface charge characteristics were assessed by attachment to either DEAE (positive)- or carboxymethyl (CM) (negative)-Sephadex ion-exchange beads. Hydrophobicity and adhesiveness of nonencapsulated, wild-type R. toruloides decreased from mid-log to late stationary phase. Encapsulated wild-type R. toruloides cells were more hydrophobic and more adhesive than nonencapsulated cells. However, two encapsulated Att− mutants were more hydrophobic than the wild type and levels of adhesion of R. toruloides were similar on polystyrene and less hydrophobic glass surfaces. Adhesion of wild-type yeast to barley and polystyrene was correlated with attachment to CM-Sephadex beads, indicating a positive cell surface charge. Sixteen Att− mutants did not exhibit a positive cell surface charge, and wild-type yeast cells that did not attach to CM-Sephadex did not adhere to either polystyrene or barley. Wild-type R. toruloides attached to CM-Sephadex beads by the poles of the cells...

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Wang, L H; Hartman, P A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1976 EN
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Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM...

Localization and Characterization of α-Glucosidase Activity in Brettanomyces lambicus

Kumara, H. M. C. Shantha; De Cort, S.; Verachtert, H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1993 EN
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Brettanomyces lambicus was isolated and identified from a typical overattenuating Belgian lambic beer and exhibited extracellular and intracellular α-glucosidase activities. Production of the intracellular enzyme was higher than production of the extracellular enzyme, and localization studies showed that the intracellular α-glucosidase is mostly soluble and partially cell wall bound. Both intracellular and extracellular enzymes were purified by ammonium sulfate precipitation, gel filtration (Sephadex G-150, Sephadex G-200, Ultrogel AcA-44), and ion-exchange chromatography (sulfopropyl-Sephadex C-50, (carboxymethyl-Sephadex C-50). The intracellular α-glucosidase exhibited optimum activity at 39°C and pH 6.2. The extracellular enzyme exhibited optimum catalytic activity at 40°C and pH 6.0. The molecular masses of purified intracellular and extracellular α-glucosidases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 72,500 and 77,250, respectively. For both enzymes there was a decrease in the rate of hydrolysis with an increase in the degree of polymerization, and both enzymes hydrolyzed dextrins isolated from lambic wort (degrees of polymerization, 3 to 9 and more than 9). The Km values for p-nitrophenyl-α-d-glucopyranoside...

Cholera Toxins: Immunogenicity of the Rabbit Ileal Loop Toxin and Related Antigens

Kaur, Jasbir; Burrows, William; Cercavski, Lora
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1969 EN
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A method of assay of immunogenic potency of the cholera gut toxin is described; it is based on the relation of dose of antigen to neutralizing antibody titer produced in the rabbit under defined conditions and allows quantification of immunogenicity as immunogenic units per milligram of protein. Evidence, based on immunogenicity and rabbit ileal loop toxicity, is presented which indicates that the positively charged fraction of liquid-culture supernatant fluid eluted in deionized water from diethylaminoethyl Sephadex, or in electrolyte from carboxymethyl Sephadex, is a complex made up of a nonantigenic toxic moiety, a nontoxic protein component which elicits the formation of toxin-neutralizing antibody, and an inactive fraction. The complex may also be dissociated in high-salt concentrations with apparent recombination of the toxic moiety with a nondialyzable constituent of peptone to give a negatively charged complex. The immunogenic component is found in nontoxic supernatant fluids of cultures grown at pH 6.5 or in media deficient in peptone. It is also present in the nontoxic fraction eluted from diethylaminoethyl Sephadex in electrolyte or in deionized water from carboxymethyl Sephadex. When separated from the positively charged toxic moiety...

Isolation of mitogenic and adjuvant active fractions from various species of Nocardiae.

Ciorbaru, R; Adam, A; Petit, J F; Lederer, E; Bona, C; Chedid, L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1975 EN
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Delipidated lysozyme digests of Nocardia opaca, N. corallina, and N. rubra have been fractionated by Sephadex filtration. The mitogenic and adjuvant activities of the fractions thus obtained have been investigated. All fractions are mitogenic except the last fraction of N.rubra, but the N. opaca products induce a stronger stimulation of mouse spleen lymphocytes than the corresponding fractions of the two other species. The activity of the first Sephadex fractions of each strain has been compared to other mitogens (concanavalin A, lipopolysaccharide). All fractions are adjuvant, although one of them, the last Sephadex fraction of N. rubra, does not contain peptidoglycan; its activity must thus be attributed to another kind of molecule. Fractionation of the first Sephadex fraction of N. opaca by centrifugation in glacial acetic acid led to a separation of adjuvant and mitogenic activities.

Some Properties of 3-Phosphoglycerate Phosphatase from Developing Rice Grain 1

Villareal, Ruth M.; Juliano, Bienvenido O.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1977 EN
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Some properties of 3-P-glycerate phosphatase from developing caryopsis of rice (Oryza sativa L., variety IR26) were studied. The enzyme was found to be soluble and not bound to starch, and concentrated mainly in the pericarp-aleurone layer; its maximum activity was at 12 to 14 days after flowering. Contents of 3-P-glycerate and chlorophyll were highest in the grain at 7 to 8 days after flowering when starch synthesis was at a maximum. The enzyme was purified about 100-fold by precipitation with 50 to 80% ammonium sulfate, followed by chromatography through Sephadex G-200 and CM-Sephadex C-50. The pH optimum was from 5.7 to 6 and no cation was required for activity. The purified preparation had an apparent Km of 2.85 mm and was inhibited by Cu2+, Hg2+, Zn2+, Fe3+, molybdate, and F−. The enzyme also exhibited high activity toward UTP, ATP, and p-nitrophenyl phosphate; moderate activity toward other phosphates; but no activity toward phytate. A molecular weight of about 23,000 was obtained for the 3-P-glycerate peak during gel filtration on Sephadex G-200, which corresponded to a value of 26,000 for the major protein fraction by thin layer gel filtration on Sephadex G-150. Zymograms of the whole extract and semipurified preparations showed two phosphatase bands with 3-P-glycerate as substrate.

Demonstration of RNA polymerase multiplicity in Trypanosoma brucei. Characterization and purification of alpha-amanitin-resistant and -sensitive enzymes.

Earnshaw, D L; Beebee, T J; Gutteridge, W E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1987 EN
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We have isolated, characterized and substantially purified two distinct RNA polymerase activities from the flagellate protozoan parasite Trypanosoma brucei. RNA polymerases from this organism were resolved poorly on DEAE-Sephadex, but could be separated with CM-Sephadex. One form was totally resistant to alpha-amanitin, whereas the second was 50% inhibited by 10-20 micrograms of the drug/ml. The enzymes had different salt optima, but both were of high Mr (greater than 480,000) and demonstrated the template preference: poly[d(A-T)] greater than denatured DNA greater than native DNA, and both were more active with Mn2+ than with Mg2+. The amanitin-resistant enzyme, polymerase R, was partially purified by chromatography on CM-Sephadex, DEAE-Sephadex and heparin-Sepharose. This enzyme was very labile, and activity yields were around 9%; after purification, one or two protein bands could be discerned after electrophoresis under non-denaturing conditions, but about 20 polypeptides were resolved on denaturing gels, including a major component (not thought to be part of the enzyme) of Mr 65,000. Polymerase S, sensitive to low alpha-amanitin concentrations, was more extensively purified, with an 18% recovery, and yielded a single major band with two minor ones after native gel electrophoresis. Analysis under denaturing conditions permitted a possible subunit structure for this enzyme to be ascribed.

Preferential association of the insulin-like growth factors I and II with metabolically inactive and active carrier-bound complexes in serum.

Cornell, H J; Enberg, G; Herington, A C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1987 EN
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Ion-exchange chromatography of serum on DEAE-Sephadex A-50 using a stepwise NaCl gradient showed that complexes enriched with insulin-like growth factors I and II (IGF-I and IGF-II) could be preferentially eluted. A fraction eluted with 0.075 M-NaCl preferentially contained immunoreactive IGF-I with peak levels appearing in fractions of Mr approx. 110,000. The IGF-I-binding protein complex itself had low bioactivity as measured in a non-suppressible insulin-like (NSILA) bioassay. On conversion to free IGF-I by gel-permeation chromatography on Sephadex G-75 in 1% formic acid, however, the IGF-I did express its intrinsic NSILA bioactivity. In contrast, an IGF-II-enriched complex was eluted from the DEAE-Sephadex with 0.15 M-NaCl. Practically all of the recovered NSILA of the original serum was present in this fraction, in the Mr range 70,000-300,000 with a peak of 150,000. Chromatography on Sephadex G-75 in 1% formic acid separated this high-Mr NSILA into low-Mr (less than 15000) IGF-II and high-Mr acid-stable NSILA-P. The high-Mr IGF-II complex bound to concanavalin A-Sepharose, suggesting that it was a glycoprotein. The results confirm previous reports that a large portion of the NSILA of whole serum can be accounted for by a biologically active acid-dissociable complex. These data show for the first time that this active complex consists of an IGF-II-preferring binding protein. In direct contrast...

Multiple protein kinases from human lymphocytes. Identification enzymes phosphorylating exogenous histon and casein.

Kemp, B E; Froscio, M; Rogers, A; Murray, A W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1975 EN
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1. Cell-free lysates of human peripheral blood lymphocytes contained two casein kinase activities and two histone kinase activities, which could be separated by chromatography on DEAE-Sephadex. 2. Neither of the casein kinase activities were stimulated by cyclic AMP. The major activity was eluted from DEAE-Sephadex between 0.4 and 0.45M-KCl, had a molecular weight of approx. 130,000 (sucrose density gradients) and was stimulated by KCl (maximum 150mM). It also formed higher-molecular-weight aggregates when centrifuged in sucrose gradients containing 150mM-KCl. The minor activity was not retained by DEAE-Sephadex, had a molecular weight of approx. 50,000 and was not stimulated by KCl. 3. The major histone kinase activity was stimulated by cyclic AMP and was eluted from the DEAE-Sephadex column between 0.05 and 0.2M-KCl. The other activity was not stimulated by cyclic AMP and was insensitive to the rabbit muscle protein kinase inhibitor. 4. Evidence was obtained suggesting that the lymphocyte casein kinases were located primarily in the nuclei.

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Overdijk, B; van der Kroef, W M; Veltkamp, W A; Hooghwinkel, G J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1975 EN
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Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Purification and properties of the high-activity form of the enzyme.

Davies, R C; Neuberger, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/1979 EN
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1. The high-activity form of aminolaevulinate synthetase has been prepared from extracts of semi-anaerobically grown cells of Rhodopseudomonas spheroides, which were allowed to become activated in air. Specific activity was 130 000--170 000 nmol of aminolaevulinate/h per mg of protein at 37 degree C. 2. Enzyme fraction Ia prepared on DEAE-Sephadex was a mixture of four active enzymes, pI5.55, 5.45, 5.35 and 5.2, when prepared in either Tris or phosphate buffers and when extracts were activated by air or by cystine trisulphide. 3. The enzyme was further purified by preparative polyacrylamide-gel electrophoresis in imidazole/veronal buffer, pH 7.6, followed by gel filtration on Sephadex G-100 and concentration with DEAE-Sephadex. 4. The most active enzyme, pI 5.55, ran as a single protein band, mol.wt. 49 000, in sodium dodecyl sulphate and 2-mercaptoethanol. The apparent molecular weight under non-denaturing conditions was 62 000--68 000 on Sephadex G-100 or G-200, pH 7.5, and on polyacrylamide-gel electrophoresis, pH 8.5, at enzyme concentrations below 10 000 units/ml, i.e. less than 60 microgram of protein/ml, and the enzyme was mainly monomeric. 5. The enzyme was homogeneous by gel disc electrophoresis at pH 8.9 and 7.6, but a slightly more diffuse band of protein was obtained during electrophoresis in glycine buffer...

Characterization of a T-lymphocyte inhibitor in the serum of tumour-bearing mice.

Levy, J G; Smith, A G; Whitney, R B; McMaster, R; Kilburn, D G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1976 EN
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Sera from mice with large tumours from a variety of tissue types and sources have been shown to contain substances capable of suppressing the proliferative response of normal mouse lymphocytes to concanavalin A (Con A), bacterial endotoxin (LPS) and allogeneic cells. The present paper deals with studies on the nature of these inhibitory materials using mainly a methylcholanthrene-induced rhabdomyosarcoma in DBA/2J mice. It was found that a material responsible for inhibition of the Con A response eluted with immunoglobulins on Sephadex G-150 and eluted with monomeric immunoglobulin on Sephadex G-200. The component of tumour-bearer serum responsible for the suppression of the LPS response of normal lymphocytes eluted from Sephadex G-150 with the alpha and beta globulins and albumin (molecular weight less than 150,000). The immunoglobulin-containing serum fraction from tumour-bearing animals inhibited the mixed lymphocyte response, Con A response, and specific immune response to purified protein derivative (PPD) in allogeneic cell systems. It also inhibited the in vitro primary response of mouse cells to sheep red blood cells, and, to a lesser extent, the response to a T cell-independent antigen (DNP-dextran). The inhibitory activity continued to elute with monomeric IgG on Sephadex G-200 when columns were run in 1640 medium and adjusted to pH 2-5...

Mechanisms for eosinophil degranulation; release of the eosinophil cationic protein.

Winqvist, I; Olofsson, T; Olsson, I
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1984 EN
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Mechanisms for degranulation in human eosinophils were evaluated. Release of eosinophil cationic protein (ECP), a unique eosinophil granule constituent, was measured upon exposure of purified eosinophils to a large surface consisting of Sephadex beads coated with serum, which leads to complement activation. Extracellular release of approximately 15% of the cellular ECP occurred both with eosinophils from patients with eosinophilia and normal people. Almost all eosinophils isolated from patients with eosinophilia and normal people adhered to serum-treated Sephadex. The data suggest that interaction through C3 receptors is a prerequisite for ECP release from eosinophils when exposed to serum-treated Sephadex. Both cytochalasin B, cytochalasin D and hydrocortisone reduced the release of ECP. Neither the cytochalasins nor hydrocortisone inhibited the adherence of eosinophils to the Sephadex beads. Thus the inhibitory effect of these agents on ECP release is a direct effect on the degranulation process. ECF-A, histamine and colchicine did not affect the release mechanism. No direct relationship was found between degranulation and oxidative burst inasmuch as some soluble mediators induced a high respiratory burst without a concomitant ECP release. Our data suggest that mechanisms for degranulation are not fully identical in eosinophils and neutrophils.

Studies into the occurrence of soluble antigen–antibody complexes in disease. II. Criteria for distinguishing soluble complexes from other macromolecular histamine releasers

Broder, I.; Baumal, R.; Keystone, E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1968 EN
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We have defined criteria for distinguishing several macromolecular histamine releasers derived from serum. The agents characterized were aggregated γ-globulin (AGG), soluble antigen–antibody complexes (SC), antibody to γ-globulin (anti-CII) and anaphylatoxin. The criteria used included the solubility of these materials in ammonium sulphate, their behaviour on Sephadex G-200 both at pH 8·0 and at pH 3·0, their action on the guinea-pig ileum and the influence of both reduction and alkylation and of γ-globulin on their histamine-releasing activity. It was found that AGG differed from SC in being excluded from Sephadex G-200 at pH 3·0. Anti-CII could be distinguished from AGG and SC since it was partially and irreversibly inhibited by γ-globulin and was eluted in the Sephadex middle fraction at pH 8·0. Anaphylatoxin differed from AGG, SC and anti-CII in showing no inhibition by γ-globulin, in being partially soluble in 50% ammonium sulphate, in being labile on Sephadex G-200 and in producing a contraction of the guinea-pig ileum. These criteria provided a basis for identifying unknown macromolecular histamine releasers found in serum.

Direct evidence for granuloma-inducing activity of interleukin-1. Induction of experimental pulmonary granuloma formation in mice by interleukin-1-coupled beads.

Kasahara, K.; Kobayashi, K.; Shikama, Y.; Yoneya, I.; Soezima, K.; Ide, H.; Takahashi, T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1988 EN
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Pulmonary granulomas were induced in BALB/c mice by the intratracheal injection of Sephadex G-50 and latex beads. Very large granulomas developed around Sephadex G-50 beads. Minimal inflammation was produced in mice given latex beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in mice by Sephadex G-50 beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) activity. IL-1 activity in the extracts correlated with granuloma size. In a subsequent step, large granulomas were induced by the intratracheal injection of Sepharose 4B beads coupled to fractions of the extracts containing IL-1 activity (ie, granuloma-derived IL-1) prepared from Sephadex G-50-induced granulomatous lungs. In addition, large granulomas were induced by the intratracheal injection of recombinant IL-1-coated Sepharose 4B beads. In contrast, very small granulomas were seen when IL-2-coated or plain Sepharose 4B beads were injected into mice. These results indicate that IL-1 participates in the induction and/or expression of granulomas.

One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 08/04/2013 EN
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Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus...

Caracterização funcional e estrutural de novas proteases isoladas da peçonha de Bothrops alternatus e do látex de Euphorbia milii var. hislopii

Costa, Júnia de Oliveira
Fonte: Universidade Federal de Uberlândia Publicador: Universidade Federal de Uberlândia
Tipo: Tese de Doutorado
POR
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CAPÍTULO II: Uma serinoprotease da peçonha de Bothrops alternatus foi isolada por meio de três etapas cromatográficas: DEAE Sephacel, Sephadex G-75 e Benzamidina-Sepharose. A enzima purificada, denominada balternina, apresentou uma única banda protéica quando analisada em SDS-PAGE, eletroforese em gel de poliacrilamida sob condições desnaturantes, apresentando peso molecular de aproximadamente 31.500 e 27.000 na presença e na ausência de agente redutor, respectivamente. O cDNA completo foi obtido por RT-PCR e a proteína madura de 236 resíduos de aminoácidos foi codificada por 708 pares de bases. O alinhamento múltiplo das sequências de aminoácidos deduzidas desta enzima mostrou similaridade estrutural com outras serinoproteases de peçonhas de serpentes. A balternina apresentou atividades fibrinogenolítica e albuminolítica. Quando a protease e o fibrinogênio bovino foram incubados a 37 °C, na proporção de 1:10 (m/m), a enzima clivou preferencialmente a cadeia Aα e, aparentemente, não degradou as cadeias Bβ e do substrato. Testes de estabilidade mostraram que o intervalo ótimo de temperatura e pH para a atividade proteolítica sobre o fibrinogênio foram de 30 a 40 °C e 7,0 a 8,0, respectivamente. O efeito de inibidores enzimáticos mostrou que a benzamidina inibiu a atividade fibrinogenolítica...

Neutralização de atividades biológicas das peçonhas de serpentes botrópicas pelo extrato aquoso e compostos isolados de Schizolobium parahyba (FABACEAE)

Vale, Luis Henrique Ferreira do
Fonte: Universidade Federal de Uberlândia Publicador: Universidade Federal de Uberlândia
Tipo: Dissertação
POR
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CAPÍTULO I - O extrato aquoso preparado das folhas de Schizolobium parahyba (Sp), uma planta nativa da Mata Atlântica (Brasil), foi testado para avaliar sua habilidade de inibir algumas atividades biológicas e enzimáticas induzidas pelas peçonhas brutas de Bothrops alternatus e Bothrops moojeni. Cromatografia de Sp em coluna de Sephadex LH 20 resultou em 3 frações: F1 (fração metanólica); F2 (fração metanol : água, 1:1 v/v) e F3 (fração aquosa). Estas frações foram analisadas quanto a capacidade de inibir a atividade Fibrinogenolítica das peçonhas. Sp inibiu em 100% a letalidade, a incoagulabilidade sanguínea, a atividade hemorrágica e hemolítica indireta na proporção de 1:10 (peçonha/extrato, m/m) e atividade coagulante na proporção de 1:5 (peçonha/extrato, m/m) induzidas pelas peçonhas. A atividade fibrinogenolítica das peçonhas também foi neutralizada por Sp na proporção de 1:10, resultando em proteção total da cadeia Bβ e parcial da Aα do fibrinogênio. No entanto, proporções maiores de peçonha/extrato mostraram desaparecimento de todas as cadeias do fibrinogênio numa possível precipitação causada por Sp. Os testes de interação extrato/proteínas demonstraram que em determinadas proporções de extrato/proteínas Sp precipita proteínas inespecificamente...

Aplicaci??n de la espectrofluorimetr??a en fase s??lida a la determinaci??n de hidrocarburos arom??ticos polic??clicos en aguas

Olmo Iruela, Monsalud del
Fonte: null Publicador: null
Tipo: Tese de Doutorado
SPA
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Se estudian nuevos m??todos de an??lisis de hidrocarburos arom??ticos policiclicos (hap) en aguas, mediante espectrofluorimetria en fase s??lida (efs) dado su caracter carcinogenico. Se seleccionaron 12 hap y 3 absorbentes (dos hidrofilos y uno hidrofobo) para la fijaci??n y estudio de los analitos. Se consiguen bajos limites de detecci??n y pocas interferencias. A partir del estudio de fijaci??n, longitudes de onda de los m??ximos de fluorescencia y dem??s condiciones experimentales se proponen cinco m??todos de an??lisis mediante efs: Determinaci??n de antraceno en aguas, empleando c-18 s??lice por espectrofluorimetria convencional. Determinaci??n de benzo(a)pireno fijado sobre sephadex g-25, por fluorescencia sincr??nico. Determinaci??n de la mezcla pireno y benzo(a)pireno fijados en sephadex g-25 mediante fluorescencia sincr??nica derivada. Determinaci??n simultanea de pireno, benzo(a)antraceno y benzo(a)pireno fijados sobre sephadex g-25 por fluorescencia sincr??nica y, finalmente, determinaci??n simult??nea de acenafteno, fenantreno y fluoreno, fijados sobre sephadex g-25, mediante espectrofluorimetria sincr??nica de ??ngulo variable. Los m??todos propuestos se han aplicado al an??lisis de aguas de diversa procedencia comprob??ndose la validez de los m??todos propuestos

'Beta'-1,3 glucanases, proteases e quitinases : produção, purificação e aplicação.; Beta-1,3 glucanases, protease and chitinases : production, purification and application.

Luciana Francisco Fleuri
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 03/07/2006 PT
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O presente trabalho visou o estudo da produção, purificação e aplicação de b-1,3 glucanases, proteases e quitinases. A linhagem Cellulosimicrobium cellulans 191 foi utilizada para o estudo da produção de b-1,3 glucanases e quitinases e as linhagens B26 e C. cellulans 191 foram utilizadas para a produção de proteases, em meios de cultivo contendo diferentes indutores. Foram realizados planejamentos fatorias 23, e os fatores estudados foram: pH inicial, temperatura (oC) e agitação dos frascos. No planejamento experimental para a produção de b-1,3 glucanase foi verificado maior produção da enzima (0,64 U/mL) em meio de cultivo A composto por 2,0 g/L de (NH4)2SO4; 0,2 g/L de MgSO4.7H2O e 10 g/L de parede celular de levedura em tampão fosfato 0,2 M, pH 8,5, após 24 h de fermentação, a 33oC e 200 rpm. Nos planejamentos experimentais para a produção de protease pelas linhagens B26 e 191 foi verificado maior produção da enzima em meio de cultivo B composto por 2,0 g/L de (NH4)2SO4; 0,2 g/L de MgSO4.7H2O e 80 g/L de levedura seca utilizada como indutor em tampão fosfato 0,15 M, pH 6,5, após 30 h de fermentação a 20oC e 200 rpm, sendo obtido 5,01 U/mL e 4,25 U/mL, respectivamente. No planejamento experimental para a produção de quitinase foi verificado maior produção da enzima (7...