ECTEOLA Columns Research Articles

The metabolic heterogeneity of glycosaminoglycans of the different zones of the epiphyseal growth plate and the effect of ethane-1-hydroxy-1, 1-diphosphonate (EHDP) upon glycosaminoglycan synthesis in vivo.

The synthesis of total and unextractable glycosaminoglycans of rabbit epiphyseal growth plate was studied by determination of the incorporation in vivo of 35S-sulphate into various glycosaminoglycans separated by microfractionation procedures using CPC and ECTEOLA columns. In the narrow hypertrophic zone, hyaluronic acid and chondroitin sulphate of low molecular weight were found to be unextractable with 3M guanidinium chloride for 24 h, thus being firmly bound within the tissue. While the unextractable chondroitin sulphate showed a sharply reduced synthesis rate in the hypertrophic zone compared with that in the columnar and resting zones, a rapid increase in the synthesis of extractable chondroitin sulphate toward the calcification front is indicated. At microfractionation, solubility profiles indicated that the unextractable chondroitin sulphate was of predominantly lower molecular weight and/or charge density compared to that of the whole tissue, most evident in the hypertrophic zone. The distribution pattern of incorporated 35S showed a close correspondence to the distribution of the various chondroitin sulphate fractions both for extracted and unextracted tissue. In the hypertrophic zone, higher radioactivity was found for chondroitin sulphate of predominantly low molecular weight and/or charge density in extracted compared to unextracted tissue. Low doses of EHDP caused no change of distribution of the various glycosaminoglycans but generally inhibited their synthesis.

Inhibitory effects of acid polysaccharides from sea urchin embryos in RNA synthesis in vitro

The supernatant fraction from cytoplasm in sea urchin embryos exhibited a remarkable inhibitory effect on RNA synthesis in vitro and an inhibitory substance present was mostly derived from intercellular substances. This inhibitor was attributed to the acid polysaccharides. In the course of development, the inhibitory effects of the acid polysaccharides altered as indicated in the elution profiles of Ecteola column chromatography.

Alpha, an infectious macronuclear symbiont of Paramecium aurelia.

SYNOPSIS. A spiral, rod‐ or crescent‐shaped symbiont here designated alpha, is present in the macronucleus of killer stock 562, syngen 2 of Paramecium aurelia. This stock has a cytoplasmic symbiont, kappa, as well as alpha. Lines were obtained which had only alpha, others which had only kappa, and some which had neither. It was possible to purify and separate both kinds of symbiont from homogenates of stock 562 using an ECTEOLA column. The killing action of this stock is due to kappa, not alpha. Observations on the structure of alpha with the electron microscope indicate that alpha, like the cytoplasmic symbionts in this species, is a bacterium. Alpha is never seen in the micronucleus, is rarely found in the cytoplasm, but abounds in the macronucleus. If paramecia are allowed to grow slowly after autogamy, alpha passes from the old macronuclear fragments, infects the new macronucleus, and all animals retain alpha. In exautogamous paramecia growing at maximum fission rate, however, alpha often does not infect the new macronucleus and is lost from many lines when the old macronuclear fragments disappear. In mixed cultures containing alpha‐bearing and alphafree paramecia, it has been found that alpha readily invades the macronucleus of paramecia of susceptible stocks. Homogenates of alpha‐bearing cultures are also infective. Infection is highly specific, occurring in only 6 of the 44 stocks of P. aurelia in which infection was attempted, and these 6 are all syngen 2. It is suggested that the short rod or crescent form of alpha is the reproductive form, while the elongated spiral form is probably the invasive motile form.

The glycosaminoglycans of human plasma.

A method is proposed for the measurement of glycosaminoglycans (GAG) on 5-10 ml of plasma. It is based on the adsorption of GAG on small ECTEOLA columns followed by measurement of the hexuronic acid in the NaCl eluates. Routine use of the method has indicated the presence of a GAG fraction that adsorbs readily on ECTEOLA ("free" GAG) and of another that adsorbs on it only after treatment with papain ("bound" GAG). "Free" and "bound" GAG have been measured in normal adults, normal children, and children affected by mucopolysaccharidosis type I; the results obtained are in good agreement with those previously reported in the literature.Various analyses performed on purified "free" and "bound" GAG have confirmed that chondroitin-4-sulfate is the main glycosaminoglycan of normal human plasma where it occurs both free and bound to protein and at various levels of sulfation. The presence of small amounts of heparan sulfate and keratan sulfate has also been demonstrated. Metabolic experiments performed in rabbits have indicated that plasma GAG derive from peripheral tissues and increase sharply after papain injection. In young animals the "free" GAG have a faster turnover than the "bound," possibly a reflection of active processes of remodeling and calcification. The synthesis of the "free" and "bound" GAG, as measured with (85)S-sulfate incorporation, seems to proceed at the same rate, and the hypothesis has been advanced that as a result of the action of tissue proteases, part of the "bound" GAG may be transformed into "free" GAG, the latter being immediately extruded from the tissues into the circulatory system.

Effect of ionic strength on the apparent anti-coagulant activity of bovine heparin.

THE preparation of a series of fractions1 of commercial bovine heparin with varying molecular weight and biological activity by solvent (ethanol-water) elution and by Ecteola column chromatography gave us an opportunity to investigate the role of electrostatic interactions on the anti-coagulant activity of these fractions.

Physicochemical studies of fractionated bovine heparin: I. Some dilute solution properties

Commercially prepared bovine heparin was fractionated by sequential elution from an ECTEOLA column into four discrete fractions and by solvent elution with an ethanol-water mixture into 12 fractions. Equilibrium centrifugation of the fractions in 0.5 m NaCl at pH 2.5 and interference optics revealed wide distribution of molecular weights. The fractions varied in biological activity and intrinsic viscosity. Chemical differences between fractions were minimal while the biological activity for the low molecular weight fractions were uniformly low. Inactivation with HCl, under mild conditions where biological activity was reduced fivefold, was not accompanied by reduction in intrinsic viscosity or molecular weight. A linear intrinsic viscosity-molecular weight relationship for the fractions prepared by alcohol elution can be described by the functions [ η] = 1.75 × 10 −5 M w 0.98 in 0.1 m NaCl, pH 6.5, and [ η] = 7.85 × 10 −5 M w 0.80 in 0.5 m NaCl, pH 2.5. An analogous relation for the sedimentation constant determined in 0.5 m NaCl at pH 2.5 was linear and described by s 0 = 1.2 × 10 −1 M w 0.44. The data suggest that heparin in solution under the conditions studied exhibits substantial flexibility.

Effect of kinetin on the ecteola cellulose elution profile and other properties of RNA from the excised first seedling leaves of barley

The total RNA isolated by phenol-sodium lauryl sulfate procedure from fresh barley leaves and from excised barley leaves floated for 4 days in the dark on water or on 10 mg per liter kinetin solution was compared regarding its nucleotide composition, melting curve, and elution profile on Ecteola anion exchanger. Only quantitative differences were noted in the Ecteola elution profile of RNA from the three sources. The RNA from all the three sources was also similar in the nucleotide composition and the shape of the melting curve. Quantitative differences in the amount of components (designated from “a” to “m”) of RNA from three sources were noted. The most significant difference was a 50% decrease in the amount of the principal RNA component, “K,” in leaves floated on water. The component “K” was eluted from the Ecteola column with 0.1 N NaOH and was not retarded by chromatography on Sephadex G-200. This component, which is regarded as representative of the major component of high molecular weight RNA, was preserved almost 100% in leaves floated on kinetin solution. It is suggested that kinetin, by stimulating synthesis and suppressing destruction of heavy RNA, may preserve the polyribosomes of the excised leaf and thus maintain protein synthesis, which is essential for the survival of the excised leaf.

PROPERTIES OF ROUS SARCOMA HYALURONIC ACID.

SummaryHyaluronic acid isolated from Rous sarcoma was found to have an unusual effect on the precipitation reaction with cation detergent and ECTEOLA column chromatography, and to have an apparently smaller particle size than hyaluronic acid from normal tissues. This preparation exhibited promoting effects upon the spreading of a dye in tissue and upon capillary permeability. A possible role in the invasion properties of tumors is indicated.

Primer activity of thymus deoxyribonucleic acid fractionated by ecteola column chromatography

The DNA-primer activity of Ecteola fractionated calf- and rat-thymus DNA samples was determined by a DNA-polymerase assay system. All DNA samples assayed, heated or unheated, and fractionated or unfractionated, showed some degree of DNA-primer activity. The chromatographic profiles of heated DNA were different from those of unheated DNA. Phenol and p -aminosalicylate, used in the preparation of DNA, did not alter the patterns of DNA obtained by Ecteola chromatography. Sephadex chromatography was found to be a rapid and effective method to deionize DNA solutions. DNA-primer activity was increased by an ammonium hydroxide gradient. At 4°, treatment of DNA by phenol and p -aminosalicylate, Ecteola column chromatography with a salt gradient, Sephadex column chromatography with deionized distilled water to desalt DNA, and lyophilization did not alter DNA-primer activity. The results indicate that DNA-primer activity, per se , is not specifically associated with one particular fraction of DNA.

Primer activity of thymus deoxyribonucleic acid fractionated by ecteola column chromatography

The DNA-primer activity of Ecteola fractionated calf- and rat-thymus DNA samples was determined by a DNA-polymerase assay system. All DNA samples assayed, heated or unheated, and fractionated or unfractionated, showed some degree of DNA-primer activity. The chromatographic profiles of heated DNA were different from those of unheated DNA. Phenol and p-aminosalicylate, used in the preparation of DNA, did not alter the patterns of DNA obtained by Ecteola chromatography. Sephadex chromatography was found to be a rapid and effective method to deionize DNA solutions. DNA-primer activity was increased by an ammonium hydroxide gradient. At 4°, treatment of DNA by phenol and p-aminosalicylate, Ecteola column chromatography with a salt gradient, Sephadex column chromatography with deionized distilled water to desalt DNA, and lyophilization did not alter DNA-primer activity. The results indicate that DNA-primer activity, per se, is not specifically associated with one particular fraction of DNA.

Primer activity of thymus deoxyribonucleic acid fractionated by ecteola column chromatography

The DNA-primer activity of Ecteola fractionated calf- and rat-thymus DNA samples was determined by a DNA-polymerase assay system. All DNA samples assayed, heated or unheated, and fractionated or unfractionated, showed some degree of DNA-primer activity. The chromatographic profiles of heated DNA were different from those of unheated DNA. Phenol and p -aminosalicylate, used in the preparation of DNA, did not alter the patterns of DNA obtained by Ecteola chromatography. Sephadex chromatography was found to be a rapid and effective method to deionize DNA solutions. DNA-primer activity was increased by an ammonium hydroxide gradient. At 4°, treatment of DNA by phenol and p -aminosalicylate, Ecteola column chromatography with a salt gradient, Sephadex column chromatography with deionized distilled water to desalt DNA, and lyophilization did not alter DNA-primer activity. The results indicate that DNA-primer activity, per se , is not specifically associated with one particular fraction of DNA.

Quantitation of keratan sulfate in the presence of other corneal glycosaminoglycans

Glycosaminoglycans were isolated from beef cornea and resolved by anion-exchange chromatography on Ecteola columns. Keratan sulfate was measured in the presence of other glycosaminoglycans by a cysteine-H 2SO 4 method. The interference due to other saccharides and their derivatives was investigated.

Studies on corneal polysaccharides: II. Characterization

The polysaccharides of bovine corneal stroma isolated and fractionated on ECTEOLA columns have been characterized by chemical and physico-chemical methods. It was shown that the glucosaminoglycans eluted with 0.05 M hydrochloric acid solutions containing increasing concentrations of sodium chloride had a similar chemical composition but increasing molecular weight ranging from 4200 to 19,100. Chemical analysis identified the glucosaminoglycans as keratan sulphates. The galactosamino-glycan fractions all had approximately the same degree of polymerization and a molecular weight of about 40,000. Their chemical composition showed them to contain galactosamine and hexuronic acid in approximately equivalent amounts. The fractions obtained by elution with increasing Cl − concentration showed a progressive increase in the number of negative groups per disaccharide unit. All the polysaccharide preparations contained nitrogen in excess of that expected from their respective hexosamine values.

Studies on corneal polysaccharides: I. Separation

A method for the isolation of the polysaccharides of bovine corneal stroma has been developed. A procedure based on chromatography on ECTEOLA columns readily removed the peptides from proteolytic digests of corneal tissue and made possible a nearly quantitative recovery of an essentially protein-free product. A detailed investigation of the general applicability and limitations of ECTEOLA chromatography revealed that stepwise elution with salt solutions of increasing concentration effected partial separation of the individual polysaccharides. Gel filtration and zone electrophoresis studies showed that complete fractionation was not achieved because of the charge and molecular weight polydispersity within each species. Alcohol fractionation of ECTEOLA eluates, however, obviated these difficulties and proved to be highly effective in rendering a virtually complete separation of the glucosamino-glycans and the galaotosaminoglycans. The total hexosamine content of bovine corneal stroma was found to be 1.6% of the dry tissue, and at least 90% of the hexosamine appeared to be part of the polysaccharide fractions.

Studies on fractionated heparin

An investigation of heparin fractionated by cetylpyridinium chloride precipitation has shown that variations in anticoagulant activity are related to molecular weight. Chemical analysis did not reveal any difference in the chemical composition of the individual fractions, and titration indicated that the number of ionizable acid groups per disaccharide unit is constant. The fractions were found to be electrophoretically homogeneous and had the same mobilities. Molecular weights ranged from 7600 to 11,800; the relationship between limiting viscosity number and molecular weight was calculated to be [η] = 1.58 × 10 −3 M. Chromatography on ECTEOLA columns revealed that the fractions are eluted at different ionic strengths.

Stimulation of cleavage in Arbacia eggs with desoxyribonucleic acid fractions

1. 1. The effect of DNA degradation products on the cleavage of Arbacia eggs was studied. Some experiments were performed in summer when eggs were scarce and ovaries undersized. A locally made Arbacia DNA and two commercial DNAs were used, one from thymus, fibrous and highly polymerized, the other from salmon, a powder. 2. 2. The undegraded DNAs were stimulating except the highly polymerized material which was inhibitory probably due to interference with surface activities of the eggs. 3. 3. Autoclaved salmon DNA was inhibitory presumably because of split inorganic PO 4; autoclaved Arbacia DNA was stimulating and the highly polymerized autoclaved material was inactive. 4. 4. Autoclaved DNA solutions were passed through a cellulose ECTEOLA column and fractions eluted with 0.1–2.0 M NaCl solutions (the main constituents of such fractions are polynucleotides). 5. 5. Fractions of autoclaved salmon DNA were inactive but the Arbacia DNA fractions were stimulating. 6. 6. Crystalline DNA-ase was used to digest the three DNAs and fractions of the digest were collected from ECTEOLA column as in no. 4. 7. 7. Fractions of salmon DNA digest were inactive whereas fractions of Arbacia and highly polymerized digests were very active, sometimes raising the rate of division to 100 per cent as compared with the very low summer rate of about 5 per cent. 8. 8. Differences in the actions of the three DNAs were explained on the basis of their degree of polymerization. The pronounced stimulation of cleavage of summer eggs was explained as correction of some deficiency in polynucleotides. Fractionation selected biologically active material (polynucleotides) with specific properties.

The purification and chromatography of bacteriophages on anion-exchange cellulose

A method is described for the purification of the bacteriophages T1, T2r, and T2r + from lysates of Escherichia coli using the cellulose anion-exchange adsorbent ECTEOLA. The properties of the virus preparations isolated by this method indicate that the bacteriophages are recovered in states of purity comparable with those of phage preparations obtained by other procedures. The cbromatography of live bacteriophage, virus nucleic acid, and virus protein upon ECTEOLA columns is described.