Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into
two groups based on their consensus sequences and the aptamers from
both groups showed strong binding to Sephadex G-100. One of the
highest affinity aptamers, D8, was chosen for further characterization.
Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but
not to isomaltose, isomaltotriose and isomaltotetraose,
suggesting that its optimal binding site might consist of more than
four glucose residues linked via α-1,6
linkages. The aptamer was very specific to the Sephadex matrix and
did not bind appreciably to other supporting matrices, such as Sepharose,
Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer
could be purified from a complex mixture of cellular RNA, giving
an enrichment of at least 60 000-fold, compared with a non-specific
control RNA. These RNA aptamers can be used as affinity tags for
RNAs or RNA subunits of ribonucleoproteins to allow rapid purification
from complex mixtures of RNA using only Sephadex.
Chromatography on Sephadex LH20, in a linear gradient of methanol in 0.02M TEAB buffer pH 7.5, is proposed as a fast and efficient method for the isolation and purification of protected oligoribonucleotide phosphodiesters obtained by deprotection of internucleotide phosphotriesters, and for the monitoring of the deprotection step itself. Its utility is shown on the example of removal of 2,2,2-trichloroethyl groups from oligoribonucleotide phosphotriester I of sequence CCCAUAA by two methods: /1/ reductive elimination with zinc in the presence of acetylacetone modified as presented here, and /2/ hydrogenolytic dehalogenation over palladium in pyridine. This method of chromatography on Sephadex LH20 is used as a key purification step during the removal of 2,2,2-trichloroethyl groups from I by method /1/ and allows to raise the yield of III during fianl deprotection step from 5 to 65%.
Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.
To facilitate the diagnosis of recent rubella infection, rubella haemagglutination inhibiting antibody has been determined in four fractions obtained by Sephadex G-200 gel filtration of samples of serum. All the 21 samples collected at the convalescent stage of the disease had varying proportions of haemagglutination inhibiting antibody in fraction 1, representing the major portion of IgM antibody whereas all but three out of 22 sera from persons with no history of recent rubella had negative titres in this fraction. The haemagglutination inhibiting titres in the three positive sera in the second group was very low as compared to the other fractions. Fractionation of sera on a Sephadex G-200 column coupled with the rubella haemagglutination inhibition test can, therefore, be used to diagnose recent rubella infection.
Cell extracts from Bacillus brevis (A.T.C.C. 10068), grown with various media, incorporated certain 14C-labelled amino acids that are normally components of tyrothricin into material that was extracted by ethanol from the precipitate formed by adding acid. When this material was separated by paper and silica-gel thin-layer chromatography and paper electrophoresis 14C was located in those regions that also contained gramicidin and tyrocidine. From a study of the properties of the system responsible for the incorporation it was deduced that non-tyrothricin materials were present. It was shown that the methods normally used to characterize tyrothricin do not adequately distinguish between tyrothricin and non-tyrothricin materials. However, a method for separating these materials was devised. This involved elution with ethanol from columns of acid alumina followed by gel filtration on Sephadex LH-20 with dimethylformamide–water solvent. The behaviour of gramicidin and tyrocidine on the Sephadex LH-20 column was examined, and it was concluded that the separation was not caused simply by gel filtration of unassociated molecules. Also, tyrocidine molecules with different amino acid compositions seemed to have different affinities for the Sephadex LH-20 column.
1. The measurement of osmotic pressure by means of a single bead of Sephadex (Edmond, Farquhar, Dunstone & Ogston, 1968) has been made more precise by immobilizing the bead on a fine needle. The design, calibration and use of the osmometer are described. 2. The method is particularly suitable for measuring high osmotic pressures in solutions of high-molecular-weight solutes, which must not penetrate the Sephadex to a significant extent. 3. With Sephadex G-50 the limit of precision is about 1.5cmH2O and the lower limit of molecular weight for a solute of compact molecular form is about 105. 4. The time required for each equilibration is less than 10min. 5. An impaled bead can be stored in the dry state without affecting its calibration. 6. Measurements on a sample of polyvinyl alcohol, degree of polymerization stated as 1750±50, gave ¯mn 43000±3000 and A2 (6.0±0.24)×10−4.
1. Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. Suitable choice of the gradient results in the separation from each other of oestrogen sulphates, oestrogen 16(or 17)-glucosiduronates, oestrogen 3-glucosiduronates and free oestrogens. 3. Evidence for the presence of oestriol 3-sulphate, oestrone 3-sulphate, 17β-oestradiol 3-sulphate, 16-oxo-17β-oestradiol 3-sulphate and oestriol 16(or 17)-sulphate in the sulphate fraction of DEAE-Sephadex chromatograms of pregnancy urine is provided.
1. Sephadex in bead form shows reversible changes of inner volume when immersed in solutions of a non-penetrating solute. These changes are in accordance with the theory of Flory (1953) for the swelling of gels. This makes possible the use of single beads for measuring the osmotic pressures of polymer solutions, up to or beyond 1kg./cm.2. 2. Measurements of the inner volume of Sephadex G-200 by equilibrium dilution, at various concentrations of dextran 500, gave values in agreement with those obtained from the dimensions of single beads. 3. Measurements by chromatography of the inner volume of Sephadex G-200 gave values that differed slightly but significantly from those obtained by the other methods. Similar small disagreements were found between the dilution and chromatographic values for bovine serum albumin measured in the presence of various concentrations of dextran 500. The major factor that affects the dependence of the chromatographic elution volume (of dextran 500) on concentration is shown to be the change of the inner volume of the Sephadex, with dynamic factors playing a minor role. 4. Contrary to the findings of Ackers (1964), the equilibrium dilution and chromatographic methods gave closely agreeing values for the distribution coefficient...
1. Human growth hormone was prepared from acetone-dried residues after extraction of gonadotrophins from pituitary glands. 2. Crude growth hormone was purified by gel filtration on Sephadex, resulting in a product that is soluble in water or 0·5% sodium chloride. It is painless on injection and shows a twofold increase in biological potency. Aggregation of growth hormone on Sephadex columns can be avoided by the addition of urea (6m) and EDTA (1mm) to the buffer. 3. Growth hormone appeared as a single component from Sephadex and ion-exchange columns and sedimented as a single boundary in the ultracentrifuge. In the circular disk electrophoresis, however, the growth hormone showed one faster and two slower-moving anionic components. 4. These components were isolated by preparative electrophoresis on polyacrylamide columns. The purified growth hormone and its three components sedimented as single boundaries with coefficients 2·62, 2·66, 2·66 and 2·83s respectively. 5. Amino acid analyses of the purified growth hormone and its components were closely related. End-group analysis of purified growth hormone and its components showed only phenylalanine at both N- and C-terminals. 6. The purified growth hormone and its components were essentially free of other pituitary hormones...
Tumour necrosis factor-α (TNF-α) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-α is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-α expression influences recruitment of specific inflammatory cell types.A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24–72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively.Messenger RNA encoding TNF-α was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-α protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration.Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells...
A comparison was made of the effect of DEAE (diethylaminoethyl) Sephadex (an anion exchange resin) and cholestyramine (Questran) with and without the addition of clofibrate in normal and hypercholesterolaemic patients. DEAE Sephadex (12-15 g/day) alone appeared to be as effective as cholestyramine in lowering the plasma cholesterol by 12-15%. Clofibrate acted synergistically with DEAE Sephadex and increased the activity of the latter by over twofold. This combination proved superior to that of clofibrate and cholestyramine and has the greatest potential use in the treatment of type II pattern hyperlipoproteinaemia.
The CC chemokine eotaxin is a potent and specific eosinophil chemoattractant. Eosinophil-dependent tissue injury has been shown to contribute to airway inflammation such as that in asthma. In the present study, We investigated eotaxin expression in a rat model of pulmonary inflammation (featuring accumulation of eosinophils) induced by intratracheal instillation of cross-linked dextran beads (Sephadex G200). Intratracheal instillation of 5 mg/kg Sephadex caused a time-dependent eosinophil infiltration into the lung, reaching a peak at 24 hours. Eotaxin mRNA in the lung paralleled the eosinophil influx. Eotaxin protein in bronchoalveolar (BAL) fluids and lung homogenates was shown by Western blot and immunostaining to be maximally expressed by 24 hours. Sephadex-induced lung injury, as measured by 125I-labeled albumin leakage from the pulmonary vasculature, developed in a time-dependent manner. Intravenous injection of blocking antibody to eotaxin significantly decreased eosinophil infiltration and lung permeability. These data suggest that, in the Sephadex model of lung inflammation, eotaxin up-regulation mediates intrapulmonary accumulation of eosinophils and the development of lung injury.
The effects of disturbances of the pulmonary circulation on the formation of pulmonary emphysematous bullae in rabbits were studied with the use of changes in the parenchyma of lungs insulted by both pulmonary embolization with Sephadex beads and carrageenan-induced pneumonia. Rabbits given one of the larger doses of Sephadex and then carrageenan in solution had giant bullous lesions in the lobe treated with those agents 2 months later. More animals had bullous lesions in the groups given the larger doses of Sephadex than in groups given the smallest dose or none. In animals killed after 2 weeks, bullous lesions were found, whereas there were none in those killed after 1 week. These results suggest that the size and number of bullous lesions forming in the treated lobes are associated with the dose of Sephadex. Giant and small bullous lesions are varieties of the same disorder, and there seems to be a latent period for development.
1. Rats given an intravenous injection of Sephadex particles (0.5 mg of G200 in 1 ml of saline) on days 0, 2 and 5 had a blood eosinophilia which was maximal on day 7. 2. On day 7, broncho-alveolar lavage (BAL) fluids taken from the rats contained an increased number of eosinophils and fewer mononuclear cells but there was no change in the small number of neutrophils. In addition the rats were hyper-sensitive to the increase in resistance to artificial respiration produced by 5-hydroxytryptamine (5-HT), given intravenously, with a shift to the left of the log dose-response curve. Lung parenchymal strips, taken from the rats on days 6, 7 and 8, were hyper-reactive to 5-HT with an increase in slope of the log dose-response curve. 3. Compounds with a wide variety of activities were evaluated for their effects on the blood eosinophilia on day 7 when given before each injection of Sephadex. The eosinophilia was reduced by glucocorticosteroids, beta-adrenoceptor agonists, aminophylline, dapsone and phenidone. 4. Dexamethasone, isoprenaline, dapsone and phenidone at doses that reduced the blood eosinophilia also reduced the changes in number of leucocytes in the BAL fluids and the hyper-responsiveness to 5-HT in vivo and in vitro, except that the effects of dapsone on the hyper-sensitivity to 5-HT in vivo did not reach significance. Aminophylline was the least effective of the drugs at reducing the blood eosinophilia and its effects on the other changes did not reach significance. Sodium cromoglycate reduced the BAL eosinophilia but had no effect on the other changes produced by Sephadex.(ABSTRACT TRUNCATED AT 250 WORDS)
Sephadex particles (20-80 μ in size) were injected into the abdominal aorta of 134 male Sprague-Dawley rats near the renal arteries. In 31 rats, the right kidney was then removed. The Sephadex particles lodged in glomerular capillaries, afferent glomerular arterioles and interlobular arteries, creating renal infarcts, some of which were grossly visible. Shortly after injection, arterial blood pressure rose significantly in most animals. The hypertension in uninephrectomized rats was not demonstrably different from that in rats with two Kidneys. Severity and duration of hypertension (up to 8 months) were positively correlated with the number of Sephadex particles in renal vessels, and there was also a positive correlation between the degree of hypertension and serum urea nitrogen levels, and between degree of hypertension and degree of cardiac hypertrophy. The vascular permeability in acutely hypertensive rats was abnormal, as judged from penetration of iron-dextran into vessel walls. This experimental model resembles atheromatous microembolic renovascular disease, which may play a significant role in the pathogenesis of unexplained hypertension in patients with advanced aortic atherosclerosis.
1. The effect of rapamycin (0.001 to 5 mg kg-1) on the increased leukocyte counts in bronchoalveolar lavage (BAL) fluid and hyperreactivity of isolated bronchial strips to histamine and acetylcholine (ACh) was studied following the intravenous injection of Sephadex beads to guinea-pigs. 2. The intramuscular (i.m.) injection of rapamycin (0.012 to 5 mg kg-1) dose-dependently inhibited the increase in leukocyte counts in BAL fluid. Rapamycin (5 mg kg-1) reduced the numbers of eosinophils neutrophils, macrophages and lymphocytes in BAL fluid by 64, 55, 19 and 50% respectively. In addition, rapamycin (0.012 to 5 mg kg-1) significantly inhibited the Sephadex-induced hyperreactivity of bronchial tissue to both histamine and ACh. 3. At a dose of 0.001 mg kg-1, rapamycin did not significantly reduce leukocyte infiltration or bronchial hyperreactivity. 4. Cyclosporin (5 mg kg-1) significantly reduced both lymphocyte and eosinophil numbers in BAL fluid of Sephadex-injected guinea-pigs whereas dexamethasone (1 mg kg-1) significantly reduced lymphocyte numbers. Neither drug affected the bronchial hyperreactivity to histamine and ACh. 5. It is concluded that the new immunosuppressive drug, rapamycin, is a potent inhibitor of leukocyte migration and bronchial hyperreactivity observed following the intravenous injection of Sephadex beads to guinea-pigs. Rapamycin also appears to be more effective than cyclosporin or dexamethasone in reducing leukocyte counts and bronchial hyperreactivity in this model. 6. Our results suggest that inflammatory mechanisms which are inhibited by rapamycin may be important in the induction of Sephadex-induced hyperreactivity.
The β chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo...
Hemoderivados são medicamentos produzidos pelo fracionamento industrial do plasma
humano. O principal método para purificação de proteínas plasmáticas humanas é baseado
no método de precipitação com etanol desenvolvido por Cohn-Oncley. O uso de
metodologias simples, de baixo custo e eficientes constitui um avanço tecnológico para
obtenção desses hemoderivados. O objetivo deste trabalho foi imobilizar heparina
comercial em Sephadex G-25 revestido com polianilina e posteriormente utilizar o
derivado imobilizado como matriz de afinidade para purificação de proteínas do plasma
humano. Para síntese do derivado imobilizado, Sephadex G-25 foi revestido com
polianilina e tratado com glutaraldeído, em seguida, incubado com solução de heparina
ativada com 1-etil-3-(dimetilaminopropil) carbodiimida e N-hidroxisuccinimida. A fim de
determinar a influencia de variáveis na imobilização de heparina em Sephadex-polianilina,
foi realizado planejamento experimental fatorial fracionário (24-1) no qual se avaliou quatro
variáveis independentes: concentração e tempo de reação do glutaraldeído e concentração e
tempo de reação da heparina. Nas condições otimizadas desses níveis, a heparina
imobilizada em Sephadex-polianilina foi utilizada para purificação de proteínas do plasma
humano. As variáveis concentração de glutaraldeído e concentração de heparina foram
estatisticamente significativas na imobilização de heparina ao Sephadex-PANI e foi obtido
melhor rendimento de heparina imobilizada (6...
1. We have measured the partition coefficients of bovine serum albumin with Sephadex grades G-100, G-150 and G-200, and of a dextran ([unk]n 19700) and a polyethylene glycol ([unk]n 8000) with Sephadex G-200. We have also measured the effects of these solutes on the inner volumes of the grades of Sephadex. 2. The results can be described with fair consistency by means of a simple thermodynamic treatment that makes use of the virial coefficients of Sephadex and of the solute, and of a coefficient that expresses their interaction. This coefficient is related to the `exclusion volume' of Sephadex for the solutes. 3. The Sephadex G-200–polyethylene glycol system shows anomalies of behaviour that are ascribed to the occurrence of `incompatible' phase separation within the Sephadex beads.
This work aims to compare the obtention of k-casein as to its purity, yielding and obtention rate. It was concluded that casein fractioning with urea separates k-casein with a high degree of purity. Both methods showed practically identical eletrophoretic resu1ts, while the Sephadex filtration method gives some advantages.; O presente trabalho teve como objetivo comparar a obtenção de k-caseína em termos de pureza, rendimento e rapidez de obtenção. Concluiu-se que o fracionamento da caseína por uréia separa em alto grau de pureza a K-caseína. Os dois métodos apresentaram resultados eletroforéticos praticamente iguais, sendo que o método da filtração em gel Sephadex oferece algumas vantagens.