Two Organic Fixatives for Acid Mucopolysaccharides

Cetyl pyridinium chloride, 0.5% in 4% aqueous formaldehyde and 5-aminoacridine hydrochloride, 0.4% in 50% aqueous ethanol, have been tested as fixatives for acid mucopolysaccharides in a variety of tissues. These solutions are superior to 4% aqueous formaldehyde, Carnoy's fluid and basic lead acetate for this purpose and also give good nuclear fixation. Tissues containing these mucopolysaccharides are particularly well defined with the acridine-ethanol fixative.     

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.     

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.     

The Reactions of Isolated Mucopolysaccharides to Several Histochemical Tests

The reaction to three histochemical tests of preparations of hyaluronic acid, chondroitin sulfate, heparin, the acidic mucopolysaccharides from cornea, gastric mucin, and dentine, and also of the neutral mucopolysaccharide from gastric mucin was studied. To 1% aqueous solutions of the acid mucopolysaccharides, equal volumes of 1% casein solution were added; drops of the resulting solutions were placed on slides and dried at 37 °C. The films were then fixed in acetic-alcohol (1:9). The technics employed were the periodic acid-Schiff (PAS) test, the metachromatic reaction and the Hale test. The relative acidity of the preparations was demonstrated by staining in dilute aqueous methylene blue at pH 3-6. With the exception of the preparation from dentine, the acid mucopolysaccharides stained only weakly with PAS; the neutral mucopolysaccharide stained strongly. It is concluded, therefore, that the use of the PAS technic for the histochemical demonstration of acid mucopolysaccharides is misleading, for many important members of this class of tissue component do not react appreciably. On the other hand, metachromasia was shown by all the acidic compounds studied, and the intensity of staining was approximately correlated with the acidity of the preparations. The Hale method was found to be nonspecific.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Indirect sulphonation of mucopolysaccharides and glycogen in tissue sections with the use of bisulphite or dithionite after periodic acid oxidation.

Tissue mucopolysaccharides and glycogen can be indirectly sulphated after being oxidized by periodic acid and treated with sodium bisulphite or dithionite in aqueous solution. The sulphated sites are darkly stained by Roluidine Blue and realted dyes at pH less than 1.0. The background is very pale or colourless. The stained sections resist extraction with 1% hydrochloric acid for 48 hr, but can be extracted by 5% ammonium hydroxide in ethanol in 1 hr. Other oxidizing agents cannot be substituted for periodic acid.     

Formaldehyde fixation of proteins and small polypeptides after isoelectric focusing in polyacrylamide gels

High-sensitivity detection of proteins and small polypeptides after isoelectric focusing is achieved by immersing the polyacrylamide gel directly in an aqueous Coomassie brilliant blue G-250 solution containing 40% methanol and 4% formaldehyde. The carrier ampholytes remain soluble under these conditions, whereas polypeptides over a wide range of molecular masses and isoelectric points are precipitated within the gel matrix.     

Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils.

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48--61% chondroitin 4-sulphate, 20--30% dermatan sulphate, 10--15% heparin and 7--9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.     

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.     

Experimental Cyanine Red: A New Stain for Nucleic Acids and Acid Mucopolysaccharides

A new cationic dye, experimental cyanine red (du Pont), with an absorption maximum of 536 mμ and a pH of 2.9 in 0.5% aqueous solution, is shown to be suitable for staining nucleic acids and tissue materials presumed to contain acid mucopolysaccharides. Mammalian tissues fixed in 10% neutral buffered formalin or Bouin's fluid are dehydrated, embedded in paraffin, sectioned, mounted, deparaffinized, passed through ethanols to water, and stained for 3-30 min in 0.5% experimental cyanine red in water. Differentiation and dehydration in 3 changes (about 1 min each) of n-butanol is followed by clearing in xylene and mounting in resin.     

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%.     

Sporostatic and Sporocidal Properties of Aqueous Formaldehyde

Aqueous formaldehyde is shown to exert both sporostatic and sporocidal effects on Bacillus subtilis spores. The sporostatic effect is a result of the reversible inhibition of spore germination occasioned by aqueous formaldehyde; the sporocidal effect is due to temperature-dependent inactivation of these spores in aqueous formaldehyde. The physicochemical state of formaldehyde in solution provides a framework with which to interpret both the sporostatic and sporocidal properties of aqueous formaldehyde.     

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.     

Gene conversion vs point mutation in generating variability at the antigen recognition site of major histocompatibility complex loci

We have prepared a [³²p]-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH₂) function at its 3-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.Abbreviations HBA 4-hydroxybenzoic acid, Na⁺ salt - HBzOH 2-hydroxybenzyl alcohol - HBSA 4-hydroxybenzenesulfonic acid, Na⁺ salt Correspondence to: L.E. Orgel     

New Formaldehyde Base Disinfectants

Preparations of formaldehyde in various organic liquids-ethylene glycol, glycerol, and propylene glycol-serve as effective disinfectants towards microbial vegetative cells and spores. This disinfection is a temperature-dependent process and is manifest when these formaldehyde base disinfectants are dissolved in water. The irritating vapors associated with formaldehyde disinfection are not present in either of these new formaldehyde base disinfectants or in aqueous solutions of them.     

The conversion of injected glucose into renal glycogen and mucopolysaccharides

Summary Tritiated glucose has been injected into rabbits in various states of hydration. The renal papilla of all animals showed an uptake of the label, converted into glycogen, and into mucopolysaccharides, in a manner dependent on the water balance of the animal.In papillae of control animals, the glycogen of the collecting duct epithelial cells and the mucopolysaccharides of the interstitium were labelled.In papillae of animals in an aqueous diuresis, the collecting duct glycogen was lightly labelled and there was no label over the interstitium.Antidiuretic hormone caused a diversion of label from the collecting ducts into interstitial mucopolysaccharides.The significance of these findings, with respect to renal concentrating ability, is discussed.The author wishes to thank Dr. M. K. S. Hathorn for help with the statistical analysis. Mr. K. Gamblin and Mr. P. L. Hyam gave valuable technical assistance. This research formed a part of the work approved for the degree of Ph. D. (London).     

The Relationship between Structure and Activity of Taurolin

Taurolin [Bis(1,1-dioxo-perhydro-1,2,4 thiadiazinyl-4)methane] is an antimicrobial compound formed by the condensation of two molecules of taurine with three of formaldehyde. It has been suggested that it releases formaldehyde in contact with bacteria. Evidence from TLC, HPLC and NMR spectroscopy indicates that taurolin is mostly hydrolysed in aqueous solution to release one molecule of formaldehyde and two monomeric molecules (1,1-dioxo-perhydro-1,2,4-thiadiazine and its carbinolamine derivative). A stable equilibrium is established. Antibacterial activity is not entirely due to adsorption of free formaldehyde but also to reaction with a masked (or latent) formaldehyde, as the activity of taurolin is greater than formaldehyde. The monomer is only slightly active by comparison.     

Water-stable fluorophores,produced by reaction with aldehyde solutions,for the histochemical localization of catechol- and indolethylamines

Summary The properties of a new fluorescence histochemical method for arylethylamines based on reaction with a mixture of 4% formaldehyde and 0.5% glutaraldehyde in aqueous solution are described. At room temperature the aldehyde mixture produced a well-localized fluorescence reaction in tissues, which, when examined microscopically in aqueous solution, was sufficiently intense for fine terminal noradrenergic axons to be seen. If the tissue was subsequently dried, the fluorescence intensity increased. At the same time as inducing the fluorophores, the aldehyde mixture fixed the tissue to a standard well suited for electron microscopy. It thus proved possible to locate amine containing cells in the fluorescence microscope and subsequently examine their ultrastructure. In aqueous models, the aldehyde mixture formed fluorescent products with adrenaline, noradrenaline, dopamine, dopa, 5-hydroxytryptamine and 5-hydroxytryptophan, but not with histamine or octopamine. The fluorescence induced in the aldehyde mixture remained stable if the tissue was subsequently transferred to saline or distilled water and when it was dehydrated in ethanol and cleared with xylene, benzene, chloroform or acetone.     

Water-stable fluorophores, produced by reaction with aldehyde solutions, for the histochemical localization of catechol- and indolethylamines.

The properties of a new fluorescence histochemical method for arylethylamines based on reaction with a mixture of 4% formaldehyde and 0.5% glutaraldehyde in aqueous solution are described. At room temperature the aldehyde mixture produced a well-localized fluorescence reaction in tissues, which, when examined microscopically in aqueous solution, was sufficiently intense for fine terminal noradrenergic axons to be seen. If the tissue was subsequently dried, the fluorescence intensity increased. At the same time as inducing the fluorophores, the aldehyde mixture fixed the tissue to a standard well suited for electron microscopy. It thus proved possible to locate amine containing cells in the fluorescence microscope and subsequently examine their ultrastructure. In aqueous models, the aldehyde mixture formed fluorescent products with adrenaline, noradrenaline, dopamine, dopa, 5-hydroxytryptamine and 5-hydroxytryptophan, but not with histamine or octopamine. The fluorescence induced in the aldehyde mixture remained stable if the tissue was subsequently transferred to saline or distilled water and when it was dehydrated in ethanol and cleared with xylene, benzene, chloroform or acetone.