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Polybitoxins: A group of phospholipases A2 from the venom of the neotropical social wasp paulistinha (Polybia paulista)

Oliveira, Marcia Regina de; Palma, Mario Sergio
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 189-199
ENG
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The neotropical wasp Polybia paulista is very aggressive and endemic in south-east Brazil, where it frequently causes stinging accidents. By using gel filtration on Sephadex G-200, followed by ion-exchange chromatography on DEAE-Cellulose under a pH gradient, a group of four toxins (designated as polybitoxins-I, II, lII and IV) presenting phospholipase A2 (PLA2) activities was purified. These toxins are dimeric with mol. wts ranging from 115,000 to 132,000 and formed by different subunits. The four toxins contain very high sugar contents attached to their molecules (22-43% w/w) and presented different values of pH optimum from 7.8 to 9.0; when dissociated, only residual catalytic activities were maintained. The catalytic activities of polybitoxins (from 18 to 771 μmoles/mg per minute) are lower than that of PLA2 from Apis mellifera venom and hornetin from Vespa basalis. The polybitoxins presented a non-linear steady-state kinetic behavior for the hydrolysis of phosphatidylcholine at pH 7.9, compatible with the negative co- operativity phenomena. All of the polybitoxins were very potent direct hemolysins, especially the polybitoxins-III and IV, which are as potent as the lethal toxin from V. basalis and hornetin from Vespa flavitarsus...

Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp

Bocchini, Daniela A.; Damiano, Valquiria B.; Gomes, Eleni; Da Silva, Roberto
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Conferência ou Objeto de Conferência Formato: 393-402
ENG
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The alkalophilic Bacillus circulans D1 was isolated from decayed wood. It produced high levels of extracellular cellulase-free xylanase. The enzyme was thermally stable up to 60°C, with an optimal hydrolysis temperature of 70°C. It was stable over a wide pH range (5.5-10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0. This cellulase-free xylanase preparation was used to biobleach kraft pulp. Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (κ number) and brightness, respectively. Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights.

Purificação de lacases PPO-I de Botryosphaeria rhodina

Hiroito Obara, Francis Widmann; Varéa-Pereira, Geni; Tomoe Miyagui, Dalva; Corradi Da Silva, Maria de Lourdes
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 303-310
POR
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Laccases are glycoprotein polyphenol oxidases which are involved in fungal pathogenicity and they are also useful for biotechnological applications. The ligninolytic ascomycete, Botryosphaeria rhodina, has been studied as producer of exopolysaccharide and PPO-I and PPO-II laccases induced by veratryl alcohol. However, as the induced laccases have not been isolated, the aim of this study was to purify the enzyme and to identify the carbohydrates constituents of the glycosidic moiety. The fungus was cultivated on broth Vogel, 1% glucose and 30.4mM veratryl alcohol during 4.5 days at 28°C/180 rpm. The extracellular fluid showed high carbohydrate concentration and the stability of PPO-I laccase under conditions of refrigeration and freezing at 4°C-18°C over 40 days. The purification was developed by ultrafiltration using a NMWL 100 and 30 kDa membrane, gelfiltration on Sephadex G-100, and ion-exchange chromatography on DEAE-cellulose. The purified laccase was identified as a glycoprotein, weight molecular 113 kDa, consisting of 40% protein and 60% carbohydrate identified by HPAEC-PAD as fucose, galactose, mannose, glucose and glucosamine.

Flavonóides de Calotropis procera R. Br. (Asclepiadaceae)

Gallegos-Olea, R. S.; Borges, M. O R; Borges, A. C R; Freire, S. M F; Silveira, L. M S; Vilegas, Wagner; Rodrigues, C. M.; Oliveira, A. V.; Costa, J. L.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 29-33
POR
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In this work we report the identification of two flavonol glycosides isolated from the leaves of Calotropis procera R. Br. (Asclepiadaceae), a plant species with large occurrence in Northwest of Brazil with some applications in folk medicine. Some proved pharmacological activities in this species could be attributed to the presence of flavonol glycosides. The extraction and isolation of flavonol glycosides was carried out firstly by a liquid-liquid partition, and then by elution of n-BuOH fraction with MeOH over a Sephadex LH-20 column. The identification of flavonol glycosides isorhamnetin-3-O-rutinoside (1), and isorhamnetin-3-O-robinobioside (2), was obtained by 1H and 13C NMR, one- and two-dimensional techniques.

Participation of interleukin-5, interleukin-8 and leukotriene B4 in eosinophil accumulation in two different experimental models

Oliveira,Sandra HP; Faccioli,Lúcia H; Ferreira,Sérgio H; Cunha,Fernando Q
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/1997 EN
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There are several experimental models describing in vivo eosinophil (EO) migration, including ip injection of a large volume of saline (SAL) or Sephadex beads (SEP). The aim of this study was to investigate the mechanisms involved in the EO migration in these two models. Two consecutive injections of SAL given 48 hr apart, induced a selective recruitment of EO into peritoneal cavity of rats, which peaked 48 hr after the last injection. SEP, when injected ip, promoted EO accumulation in rats. The phenomenom was dose-related and peaked 48 hr after SEP injection. To investigate the mediators involved in this process we showed that BW A4C, MK 886 and dexamethasone (DXA) inhibited the EO migration induced by SAL and SEP. To investigate the source of the EO chemotactic factor we showed that mast cells, macrophages (MO), but not lymphocytes, incubated in vitro in presence of SAL released a factor which induced EO migration. With SEP, only mast cells release a factor that induced EO migration, which was inhibited by BW A4C, MK 886 and DXA. Furthermore, the chemotactic activity of SAL-stimulated mast cells was inhibited by antisera against IL-5 and IL-8 (interleukin). SAL-stimulated MO were only inhibited by anti-IL-8 antibodies as well SEP-stimulated mast cells. These results suggest that the EO migration induced by SAL may be dependent on resident mast cells and MO and mediated by LTB4...

Antibacterial activity tannin-rich fraction from leaves of Anacardium humile

Ferreira,Peracio Rafael Bueno; Mendes,Cíntia Sorandra Oliveira; Rodrigues,Cínthia Gracielly; Rocha,Juliane Caroline Moura; Royo,Vanessa de Andrade; Valério,Henrique Maia; Oliveira,Dario Alves de
Fonte: Universidade Federal de Santa Maria Publicador: Universidade Federal de Santa Maria
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/10/2012 EN
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In vitro tests conducted with extracts rich in tannins have identified several biological activities of this class of substance. Thus, this paper intends to evaluate the antibacterial activity of tannin-rich fraction obtained from leaf extracts of Anacardium humile A.St.-Hil. Extracts of A. humile leaves in 70% acetone were semi-purified with ethyl acetate and butanol. We quantified the total tannins of the semi-purified fractions, of the crude extract and of aqueous residues and then performed tests of the antibacterial activity of the tannins against Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212. All positive extracts underwent tannin isolation using a Sephadex LH-20 column. The tannins isolated from the samples were quantified and tested for the minimal inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The tannins from crude extracts, semi-purifications and residues showed inhibition of S. aureus growth with MIC=500 g mL-1. All tannin fractions showed MIC against all strains and MBC, except against E. faecalis. The tannin fractions from Anacardium humile have antibacterial activity against Staphylococcus aureus, Pseudomononas aeruginosa and Enterococcus faecalis and...

Use of Sephadex column chromatography in the assessment of thyroid status

Gimlette, T. M. D.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1967 EN
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A method is described for assessing thyroid status using Sephadex column chromatography of serum samples with added 125I triiodothyronine. The method is simple and inexpensive. It gave a correct diagnostic result in 97% of 258 euthyroid, 90% of 80 hyperthyroid, 78% of 59 hypothyroid, and 67% of 50 euthyroid, ill patients.

Value of gel filtration on Sephadex G-200 in the analysis of blood group antibodies

Stratton, F.; Smith, D. S.; Rawlinson, V. I.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1968 EN
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Analysis of blood group antibodies on Sephadex G-200 columns is described.

The separation of epinephrine from norepinephrine and dopamine from DOPA on Sephadex G-10

McDonald, T. J.; Murphy, B. E. P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1973 EN
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Epinephrine and norepinephrine were separated by acid elution through a Sephadex G-10 column with high recovery (better than 90%), excellent reproducibility, and little overlap (less than 10%). Once packed, the columns could be re-used indefinitely. Total elution time was about six hours and the columns could be left untended since the gravity flow stops automatically once the level of the eluant reaches the gel bed. The resulting dilution was five-fold. 3,4-Dihydroxyphenylalanine (DOPA) was completely separated from 3,4-dihydroxyphenylethylamine (dopamine) by the same procedure.

Serum folic acid activity (L. casei) in Sephadex gel chromatography

Markkanen, T.; Pajula, R.-L.; Himanen, P.; Virtanen, S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1973 EN
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The folic acid activity (FAA) of the protein zone in DEAE Sephadex A-50 chromatography of normal serum showed the following distribution: most (about 40%) was eluted with protein zone IV (alpha-2-macroglobulin), about 33% with protein zones II-III (transferrin), and the balance with zone V (albumin). In healthy human subjects the variations were relatively small. The repeatability of the chromatography in an individual was good.

Evaluation of new kits for the assessment in vitro of thyroid function by determination of serum total thyroxine, free TBG capacity, and free thyroxine index using Sephadex G-25 and 125I-labelled triiodothyronine and thyroxine

Howorth, P. J. N.; McKerron, C. G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1974 EN
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A trial was carried out on 134 patients of new kits (Ames Co) using columns of Sephadex G-25 for the determination of serum total thyroxine (Tetralute test) and for the indirect estimation of serum free thyroxine-binding globulin capacity (Trilute test). Both new methods were quicker and easier than the reference resin methods and of similar precision. The two measurements when combined to give a free thyroxine index (Trilute-Tetralute-FTI) increased further the diagnostic discrimination and usefulness of the tests.

Microbicidal and cytotoxic properties of soluble components from quaternary aminoethyl-sephadex.

Selsted, M E; Martinez, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1980 EN
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Soluble material released during hydration and equilibration of a quaternary aminoethyl-Sephadex anion exchanger was found to be toxic for a variety of cell types. Among these were gram-positive and gram-negative bacteria, fungi, and a murine cell line. The material was also found to interfere with the spectrophotometric enzymatic determination of lysozyme.

Principles of a competitive binding assay for the study of the interactions of poorly water-soluble ligands with their soluble binding partners. Application to bilirubin with the use of Sephadex G-10 as a competitive adsorbent.

Meuwissen, J A; Kinnaert, M; Michiels, G; Heirwegh, K P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/01/1988 EN
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1. A generally applicable method is described for obtaining experimental data on the interactions between a poorly water-soluble ligand and soluble binding factors, with the use of chemically inert solid adsorbent. The equilibrium distribution of the ligand between the liquid phase containing the soluble binders and the adsorbent must be measured and knowledge of the binding isotherm of the adsorbent is required. Procedures are given for the calculation of the binding parameters. 2. The method has been applied to quantify the interactions of bilirubin with serum and liver cytosol from the rat, Sephadex G-10 serving as the competing adsorbent. Reversible adsorption keeps the concentration of the free ligand low, thereby preventing formation of colloidal bilirubin. The sensitivity of the procedure accommodates the rather high binding affinities by which bilirubin generally interacts with its specific binding proteins. 3. The binding activities of serum and liver cytosol are of comparable magnitude. Binding of bilirubin by rat serum can be described by two independent binding sites, the affinities of which differ by two orders of magnitude. Only the site with the higher affinity appears to be of physiological importance. The major bilirubin-binding sites of rat liver cytosol seem to contribute equally to the overall binding activity of this preparation...

Heat inactivation and Sephadex chromatography of the small-intestine disaccharidases of the chick

Siddons, R. C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1970 EN
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1. The maltase, sucrase, isomaltase and palatinase activities of the chick small intestine are localized in particles that sediment when centrifuged at 100000g for 90min. 2. Solubilization of the particle-bound disaccharidases without loss of activity was achieved by digestion with papain. Trypsin was less effective and caused a preferential solubilization of the sucrase, isomaltase and palatinase activities. 3. On Sephadex G-200 columns, the solubilized preparations yielded two disaccharidase peaks. The first peak was eluted close to the void volume of the column and contained all the sucrase, isomaltase and palatinase activities and some of the maltase activity. The remainder of the maltase activity was eluted beyond the total volume of the column. 4. Precipitation with ethanol did not affect the behaviour of the disaccharidases of gel filtration. 5. The maltase activity of the second peak on rechromatography in a buffer containing 0.01m-maltose was eluted close to the void volume. 6. Similar pH optima but different Km values were obtained for the maltase activities of the two peaks. 7. Heat-inactivation studies showed that the first peak contained two disaccharidase enzymes; one hydrolysed sucrose and maltose and the other hydrolysed isomaltose...

Carp myogens of white and red muscles. Gross isolation on sephadex columns of the low-molecular-weight components and examination of their participation in anaerobic glycogenolysis

Pechère, J.-F.; Focant, B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1965 EN
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1. The combined low-molecular-weight protein components of the myogens from carp white and red muscles [about 30% (w/w) of the myogen proteins] have been isolated by gel filtration on Sephadex G-75 columns. 2. The presence in this fraction from myogen of white muscle of the three main electrophoretic components previously isolated has been confirmed, and the low molecular weight of the fourth component has been definitely established. 3. The exclusive presence of this fourth component in the myogen of red muscle, apart from myoglobin, has also been demonstrated. 4. Glycogenolysis experiments in vitro have shown that the low-molecular-weight protein fraction from carp myogen does not contain enzymes from the Embden–Meyerhof chain.

Improvement of NaNO2-Oxidizing Activity in Nitrobacter vulgaris by Coentrapment in Polyacrylamide Containing Polydimethylsiloxane Copolymer and DEAE-Sephadex

Zhang, Songping; Norrlöw, Olof; Dey, Estera Szwajcer
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2005 EN
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Removal of nitrite and nitrate from drinking water has attracted great attention in recent years because of the human health risk induced by the exposure to contaminated groundwater and surface water. We have therefore tested a model nitrite oxidation system by coentrapping the NaNO2 oxidizer Nitrobacter vulgaris with polydimethylsiloxane (PDMS) copolymer and DEAE-Sephadex in a polyacrylamide gel. The copolymer and the anion exchanger facilitate the diffusion of oxygen and NaNO2, respectively, into the gel matrix. To test the nitrite-oxidizing activity, the entrapped cells were coupled to a thermal sensor. Coentrapment of 5% (wt/vol) DEAE-Sephadex with Nitrobacter vulgaris increased the nitrite-oxidizing activity by a factor of 3.7 compared to entrapped cells alone, and by the addition of 0.86% (wt/vol) artificial oxygen carrier PDMS copolymer increased the activity further to 4.3 times higher. Operational and storage stability of the coentrapped N.vulgaris also improved. This suggests that this enhanced immobilized cell system can also be used for nitrite oxidation to nitrate in drinking water as an on-line thermally monitored bioreactor.

Poly(ethylene glycol)-induced shrinkage of Sephadex gel. A model system for quantitative analysis of osmoelastic coupling.

Ito, T; Yamazaki, M; Ohnishi, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1989 EN
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Shrinkage of Sephadex gels caused by addition of a high-molecular weight molecule, poly(ethylene glycol) (PEG) was studied. A quantitative analysis based on the cross-linked network theory by Flory and Tanaka (Tanaka, T. 1978. Phys. Rev. Lett. 40:820-823) showed that the shrinkage is due to a mechanochemical coupling between the elasticity of the network and the osmotic stress arising from preferential exclusion of PEG. These results may provide good evidence for "osmoelastic coupling", the coupling between elasticity of macromolecular structures and osmotic stress, which has been predicted in some biological systems such as phospholipid bilayer membranes (Ito, T., M. Yamazaki, and S. Ohnishi. 1989. Biochemistry. 28:5626-5630; Yamazaki, M., S. Ohnishi, and T. Ito. 1989. Biochemistry. 28:3710-3715) or actin filaments.

Clinical evaluation of Sephadex gel filtration in estimation of bilirubin binding in serum in neonatal jaundice

Kapitulnik, J.; Valaes, T.; Kaufmann, N. A.; Blondheim, S. H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1974 EN
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Clinical experience with a Sephadex gel filtration test-kit for the evaluation of bilirubin binding affinity of serum in neonatal jaundice is reported. In serial serum specimens from 166 jaundiced neonates the results of the test in most cases were in accord with the independent clinical decision to perform exchange transfusion. In all 9 cases with clinical kernicterus the Sephadex test was strongly positive (very low bilirubin binding affinity of serum).

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

Lee, Hae-Lee; Kim, Sue-Hee; Ji, Dong-Beom; Kim, Yong-Jun
Fonte: The Korean Society of Veterinary Science Publicador: The Korean Society of Veterinary Science
Tipo: Artigo de Revista Científica
EN
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The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular...

Application of Sephadex LH-20 for Microdetermination of Dopamine by Solid Phase Spectrophotometry

Taghdiri, Mehdi; Mohamadipour-taziyan, Arash
Fonte: International Scholarly Research Network Publicador: International Scholarly Research Network
Tipo: Artigo de Revista Científica
Publicado em 28/11/2012 EN
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A sensitive spectrophotometric method for the determination of dopamine was carried out without any separation steps. Bromocresol green is adsorbed on Sephadex LH-20 gel but the sorption decreases in the presence of dopamine due to ion-pair formation between bromocresol green and dopamine in solution. This attenuation was used to the microdetermination of dopamine by measurement of absorbance of the solid phase (Sephadex LH-20 gel) in a 1.0 mm cell at 625 nm. Dopamine could be determined in the concentration range of 0.4–1.6 μg mL−1 (10-mL Sample volume) with a relative standard deviation (RSD) of 0.03% (n = 4). The detection limit was obtained, 0.26 μg mL−1 (1.7 μM). The method was used for determination of dopamine in pharmaceutical injection sample and satisfactory result was obtained.