Separation of hyaluronate, chondroitin sulfate, and heparin by adsorption-desorption technique

An interesting method for separation of the three important mucopolysaccharides, hyaluronate, chondroitin sulfate, and heparin by adsorption to and elution from three inorganic salts Ca₃(PO₄)₂, BaSO₄, and Al₂O₃ has been described in details. Alumina has to be washed with dilute HCl before it can adsorb the mucopolysaccharides, and on treating with alkalies the mucopolysaccharides can be desorbed from it. Calcium phosphate and barium sulfate can adsorb the mucopolysaccharides without any pretreatment. The specific eluents for each of the polysaccharides depend on the nature of the adsorbants also. The recoveries of the mucopolysaccharides are quite satisfactory.     

Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation and fusion

The amount of hexosamines and acid mucopolysaccharides present in the rat secondary palate increases during the critical stages of palatogenesis, namely, rotation and fusion. The synthesis of acid mucopolysaccharides in vivo and in vitro in the palate was determined by the incorporation of ³H-glucosamine and Na₂S³⁵O₄. The labeled mucopolysaccharides were isolated by DEAE-cellulose chromatography and were identified on the basis of several criteria as hyaluronic acid and sulfated acid mucopolysaccharides. Hyaluronic acid accounted for approximately 60% of the total acid mucopolysaccharides synthesized in the palate both in vivo and in vitro. DON (6-diazo-5-oxonorleucine), a known inhibitor of acid mucopolysaccharide synthesis, inhibited the incorporation of ³H-glucosamine and Na₂S³⁵O₄ by palatal shelves in vitro by 70%.     

Tissue specific distribution of sulfated mucopolysaccharides in mammals.

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied. The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

Tissue specific distribution of sulfated mucopolysaccharides in mammals

A comparative study on the distribution of sulfated mucopolysaccharides in several tissues of five mammalian species is reported. It is shown that each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A/C, chondroitin sulfate B and heparan sulfate. It is also shown that the same tissue from different mammals has the same types and proportions of sulfated mucopolysaccharides, but with different molecular weights. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the five mammals studied.The possible involvement of the sulfated mucopolysaccharides in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.     

Continuous fractionation of proteins and mucopolysaccharides by simultaneous two-dimensional gel filtration and electrophoresis

A method for continuous-loading Sephadex gel electrophoresis is described. Albumin, α₁, α₂, β and γ globulins, and two kininases and an amino-peptidase are completely separated and obtained at the preparative scale by this procedure. Heparan sulfates and abnormal mucopolysaccharides from the urine of patients with Hurler's syndrome were also fractionated by this method.     

Quantal basis of vesicle growth and information content, a unified approach

Secretory vesicles express a periodic multimodal size distribution. The successive modes are integral multiples of the smallest mode (G₁). The vesicle content ranges from macromolecules (proteins, mucopolysaccharides and hormones) to low molecular weight molecules (neurotransmitters). A steady-state model has been developed to emulate a mechanism for the introduction of vesicles of monomer size, which grow by a unit addition mechanism, G₁+G_n→G_n+1 which, at a later stage are eliminated from the system. We describe a model of growth and elimination transition rates which adequately illustrates the distributions of vesicle population size at steady-state and upon elimination. Consequently, prediction of normal behavior and pathological perturbations is feasible. Careful analysis of spontaneous secretion, as compared to short burst-induced secretion, suggests that the basic character-code for reliable communication should be within a range of only 8-10 vesicles’ burst which may serve as a yes/no message.     

Interactions of sulfated mucopolysaccharides with lectins. Application to the separation of mucopolysaccharide mixtures.

The interaction of sulfated mucopolysaccharides and lectins has been studied by determining the amount of precipitate formed when mucopolysaccharides are added to a solution of concanavalin A or a partially purified lectin preparation from red kidney bean (Phaseolus vulgaris). The amount of insoluble complex obtained when a given mucopolysaccharide is added to a solution of partially purified red kidney bean preparation is pH dependent. The reaction of concanavalin A and heparin has also been studied by adding increasing amounts of mucopolysaccharide to a fixed amount of lectin. This interaction results in the development of a precipitin-like curve and leads to the isolation of a heparin fraction which has been found to be more reactive with respect to formation of a precipitate than the original heparin preparation. Monosaccharides such as α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine which are known to bind specifically to the lectin, greatly inhibit precipitate formation. The interactions between sulfated mucopolysaccharides and lectins have been used to isolate various sulfated mucopolysaccharides.     

Isolation of Lipoprotein-Acid Mucopolysaccharide Complexes from Fatty Streaks of Human Aorta

A method is presented for isolation of 1ipoprotein-acid mucopolysaccharide complexes from fatty streaks of human aorta. The complexes were extracted from fatty streaks with 0.15M NaCl and fractionated by gel filtration followed by ultracentrifugation at densities 1.006 and 1.065- The solvent density 1.065 was achieved by using D₂O instead of NaCl which decreased dissociation of the complexes. These studies demonstrated that low and very low density lipoproteins are directly involved in the complexing of lipoproteins with mucopolysaccharides in the matrix of aorta intima.     

The molecular-weight range of mucosal-heparin preparations.

A recently reported method describes the determination of the molecular-weight range of heparins by using an electrofocusing procedure to isolate individual molecular species. Commercially available heparins were fractionated on a column of polyacrylamide-agarose gel to give fractions whose molecular weights were estimated by viscometry. Fractions with mutually exclusive molecllar-weight ranges gave an appreciable number of common bands when subjected to the electrofocusing procedure; therefore, each of these bands cannot be formed from a single molecular species of heparin. Other mucopolysaccharides also gave band sequences indistinguishable from those of heparin; they differed in position and intensity with different ampholyte batches, and probably arose from individual molecular species of the ampholyte rather than the mucopolysaccharide. The molecular-weight range of the heparin was observed to be broader than that usually reported.     

Electrofocusing of heparin: fractionation of heparin into 21 components distinguishable from other acidic mucopolysaccharides.

Twenty-one fractions have been demonstrated in each of 15 different commercially available heparins subjected to electrofocusing. These fractions show a molecular-weight range from 3000 to 37,500 with a constant interval between molecular weights. Degradation of each fraction by purified enzymes of Flavobacterium heparinum yielded identical end products, suggesting chemical identity. Only fractions with a molecular weight of 7000 and up had significant anticoagulant activities. The phenomenon of electrofocusing of mucopolysaccharides is dependent upon pH, molecular weight, and ampholyte availability. Chemical composition of the mucopolysaccharide is also an essential factor since N- and O-desulfation of heparin markedly changed the focalization pattern. The pattern produced when heparin is subjected to electrofocusing is not duplicated by any other naturally occurring acidic mucopolysaccharide tested. Heparitin sulfate D shows some similarities to heparin and it is probable that heparitin sulfate D is a normal contaminant of heparin preparations (this assumption is supported by molecular-weight and anticoagulant activity determinations). The technique is specific and reproducible and unequivocally distinguishes heparin from other acid mucopolysaccharides.     

Synthesis of RNA,Protein, Cellulose,and Mucopolysaccharide and Changes in the Chemical Composition of Hartmannella culbertsoni During Encystment under Axenic Conditions*

Hartmannella culbertsoni trophozoites are transformed into viable cysts on exposure to a non-nutrient agar medium containing 15 mM MgCl₂ and 20 mM taurine. Amebae differentiating in this encystment medium incorporate more uracil-2-¹⁴C into RNA and more leucine-1-¹⁴C or valine-1-¹⁴C into proteins than controls. Encysting organisms incorporate significantly more glucose-U-¹⁴C into cellulose and glucosamine-1-¹⁴C into mucopolysaccharides. Incorporation of glucose-U-¹⁴C into cellulose and of glucosamine-1-¹⁴C into mucopolysaccharides are inhibited by actinomycin D or cycloheximide.     

Placentation in the garter snake, Thamnophis sirtalis

The structure and polysaccharide constitution of the jelly capsule of the egg of Rana pipiens is described. Microscopic examination of the jelly capsule revealed the presence of five discrete jelly layers that differed clearly in their response to selected cytochemical tests. These layers were classified as M1-through M5 from the inner to the outermost layer. A sixth layer occasionally could be observed between M3 and M4. All layers contain neutral mucopolysaccharides. In addition layers M1 and M3 contain sulphated mucopolysaccharides, M2 and M4 contain non-sulphated acid mucopolysaccharides, and layer M5 contains both sulphated and non-sulphated acid mucopolysaccharides. M2 may also contain a small quantity of sulphated mucopolysaccharides. The layer that occasionally appears between M3 and M4 is probably an area in which free acidic groups are in higher concentration than in adjacent areas rather than being a discrete jelly layer. Neither hyaluronic acid nor sialic acid was localized by the methods employed. The possible significance of some of these constituents is discussed.     

Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems

A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of mucopolysaccharides as their quaternary ammonium detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharides is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides.     

Macromolecular desorientation in detached tendons

Conclusions Based on the results obtained on the grounds of the present discussion we may conclude: Under the described condition, in the detached tendons of guinea pigs and also in the implanted tendons the acid mucopolysaccharides gradually undergo a structural disorder, loosing its molecular orientation. In the referred tendons, the loss of structural orientation of the acid mucopolysaccharides proceeds the desorientation of the collagen polypeptide chains. In the tendon repair process, under the described conditions, acid mucopolysaccharides with oriented macromolecules are elaborated during the fibrillogenesis phenomenon.This paper is part of the author's Doctoral and Livre Docencia thesis published in October 1964.Professor do Departamento de Patologia da Faculdade de Farmácia e Odontologia de Piracicaba — Govêrno do Estado de São Paulo.     

Fractionation and properties of four heparitin sulfates from beef lung tissue: Isolation and partial characterization of a homogeneous species of heparitin sulfate

Several commerical batches of heparitin sulfate extracted from beef lung tissue were fractionated into at least four distinct mucopolysaccharides by a combination of polyacrylamide and agarose gel electrophoresis. The four heparitin sulfates (A, B, C and D) were distinguished from each other and from heparin by several physical and chemical properties such as electrophoretic migration, molecular weight, presence of N-acetyl, N- and )-sulfate residues, optical rotation and enzymatic degradation. Of particular significance was the isolation of a heparitin sulfate (heparitin sulfate C) with a homogeneous molecular weight.     

Fractionation of mucopolysaccharides by countercurrent distribution in aqueous polymer two-phase systems.

A new procedure for the fractionation of mucopolysaccharides based upon differences in their partition behavior in aqueous polymer two-phase systems has been devised. Systems containing dextran, poly(ethylene glycol), trimethylamino-poly(ethylene glycol), potassium bromide and sodium phosphate buffer were employed. Countercurrent distributions were performed with a miniature countercurrent distribution device designed especially for use with aqueous polymer two-phase systems. An advantage over the widely used procedures involving precipitation of mucopolysaccharides as their quaternary ammonium detergent complexes is that the countercurrent distribution pattern of a particular mucopolysaccharide is not affected by the simultaneous presence of other mucopolysaccharides. Preliminary distributions of labelled mucopolysaccharides isolated from the cells and culture medium of monolayer cultures of rat tumor cells demonstrate that the procedure is particularly well suited for the fractionation of very minute quantities of mucopolysaccharides.     

Combined histochemical staining of 1,2-glycol and sulfate groupings of mucopolysaccharides in paraffin sections

Summary A staining procedure has been developed for the simultaneous demonstration of 1.2-glycol and sulfate groupings of mucopolysaccharides in paraffin sections. It is a combined periodic acid-Schiff (PAS) and acriflavine technique, and by this technique the different groupings of mucopolysaccharides in several tissues of the rat and mouse have been stained. The results of a series of histochemical experiments on the dual staining reaction of the mucopolysaccharides have substantiated the reliability of the present technique.     

Electron microscopical demonstration of sulphated mucopolysaccharides in mouse tracheal cartilage with a diaminobenzidine-osmium tetroxide technique

Synopsis A diaminobenzidine-osmium tetroxide method for the demonstration of sulphated mucopolysaccharides has been tested at the electron microscopical level. The reaction (which involves treatment with diaminobenzidine in an acidic solution followed by oxidation with osmium tetroxide) has been carried out directly on ultra-thin sections of mouse tracheal cartilage embedded in water-soluble glycolmethacrylate. Sulphated mucopolysaccharides in the cartilage matrix were localized as a heavy, electron-dense precipitate. The method can be applied directly to sections, overcoming in this way the difficulties of penetration, and it seems to possess a higher specificity for sulphated mucopolysaccharides than that displayed by other techniques proposed previously for the ultrastructural demonstration of acid mucopolysaccharides.     

A study of acid mucopolysaccharides in cold microtome sections of normal and experimentally hydrated bovine corneas

Summary Acid mucopolysaccharides were investigated in cold microtome sections of normal and experimentally hydrated bovine corneas. Staining methods using cationic dyes were used for the detection.A 10 min fixation of cold microtome sections in absolute alcohol did not change the stainability of acid mucopolysaccharides substantially. The staining was only a little fainter (as against unfixed sections). After 10 min fixation with formol-cetylpyridinium chloride the staining of sections was diminished and after 30 min fixation in this fluid completely abolished. After formol-calcium chloride fixative the staining was decreased in dependence on the time of fixation due to the elution of acid mucopolysaccharides in the fixative (acid mucopolysaccharides in the fixative were demonstrated by means of paper electrophoresis). Formolcalcium chloride is likewise unsuitable.Experimental hydration of corneas in distilled water did not substantially alter the staining properties of acid mucopolysaccharides in cold microtome sections. Only quantitative differences were found in comparison with untreated corneas. These differences were due to hydration causing an increase in the distance of acidic groups among individual molecules of acid mucopolysaccharides.     

Characterization of the chondroitin sulfate produced by B16 mouse melanoma cells

The mucopolysaccharides produced by B16 mouse melanoma cells have been isolated in milligram quantities from the spent media in which the cells were grown in the presence of 2-amino-2-deoxy-d-glucose-t and ³⁵S]-sulfate. The mucopolysaccharides obtained by precipitation with cetylpyridinium chloride from the Pronase digest of the media were further purified by gel filtration, ion-exchange chromatography, and treatment with nucleases. The major components were identified as chondroitin-4-sulfates by identification of the hexosamine as 2-amino-2-deoxy-d-galactose, and by digestibility with hyaluronidases, chondroitinase AC, and chondro-4-sulfatase. The o.r.d. curve and i.r. spectra of these components also confirmed their similarity to chondroitin-4-sulfate from cartilage. The molecular weight of the polysaccharide chains was estimated to be in the range 90,000–120,000 by sedimentation equilibrium analysis.