The enhanced production of hyaluronic acid by cultured rat fibroblast cells treated with cyclic AMP and its dibutyryl derivative

Cells of a newly established rat fibroblast line (SEN) in culture synthesize mucopolysaccharides, which have been identified as hyaluronic acid, chondroitin-4-sulfate and heparan sulfate. Treatment of the cells with adenosine 3′:5′-cyclic monophosphate resulted in a marked stimulation of production of hyaluronic acid, but not of the other mucopolysaccharides. Treated cells also showed increased activity of hyaluronic acid synthetase, a reduction in growth rate, and morphological alteration. In addition, 5-bromodeoxyuridine was found to counteract greatly the cyclic AMP effect.     

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.     

Histochemical observations on the ovaries of some Indian bats

Ovarian follicles of the three species of bats, Pteropus giganteus giganteus, Taphozous longimanus and Megaderma lyra lyra were studied with regard to the distribution and nature of mucopolysaccharides. Oocyte contained only glycogen. Zona pellucida showed the presence of glycogen, neutral mucosubstances, sialo- and sulfomucins. Follicle cells showed the presence of glycogen and sialic acid except in megaderma lyra lyra where the follicle cells of the Graafian follicle showed the presence of sulfomucins in addition to these mucosubstances Cumulus cells contained glycogen., sulfomucin, sialomucins and hyaluronic acid. Liquor follicular showed the presence of sulfomucins, sialomucins and hyaluronic acid. Theca cells contained glycogen and sialic acid. The luteal cells contained glycogen only.     

Histochemical study of polysaccharides in the aortic arch of some birds.

The histochemical study of polysaccharides in the aortic arch of the black vulture, turkey, peking duck, goose and duck, showed that a sialo-mucopolysaccharidic complex is present in the vessel wall and consists of: sialic acid, neutral mucopolysaccharides, hyaluronic acid and Chondrotin sulfate. The concentration of mucopolysaccharides, mainly of the acidic ones, decreases from the inner portions of the media towards the adventicia.     

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%.     

Macromolecules in the receptor lymph of campaniform sensilla

The receptor lymph of campaniform sensilla on the halteres of the blowfly, Calliphora vicina, was analyzed histochemically. Acid mucopolysaccharides were demonstrated by a test for iron-binding capacity (Hale-reaction). Further characterization by enzyme treatment showed that the receptor lymph contains hyaluronic acid and/or chondroitin sulfate. Ultrahistochemical studies gave evidence for glycoproteins in the inner and outer receptor lymph space. The significance of acid mucopolysaccharides for arthropod sensilla is discussed.     

Studies on the Indian sand lobster Thenus orientalis (Lund): mucopolysaccharides of the tegumental glands.

The tegumental glands are imbedded in the connective tissue below the epithelium of oesophagus. Each gland is made up of cells which are conical in shape with their narrow ends directed towards the lumen of the gland. In the centre of each gland there is a cavity which communicates with intracellular duct. Similar glands have been found in the hind-gut region also. These glands secrete both acid and neutral mucopolysaccharides and to some extent glycoproteins. The glands are charged mostly with the task of secreting weakly acidic mucopolysaccharides and neutral mucopolysaccharides which are confined to the apices and central cavity of the gland. The acidic nature is partly due to sialic acid and partly due to hyaluronic acid. These weak acids do not seem to play any role in digestion but lubricate the lumen of the oesophagus for easy passage of food and keep the lining of the oesophagus slimy. In the hind-gut they help in binding the faecal matter into pellets.     

Effect of hydrocortisone on the fractional makeup of acid mucopolysaccharides in the aorta]

Five components of the acid mucopolysaccharides were isolated from rabbit aorta; these were hyaluronic acid, heparitin sulphate, chondroitin sulphate A, C and B. Prolonged administration of hydrocortisone increased the hyaluronic acid concentrations and decreased the level of heparitin sulphate. Fifteen days after the cessation of the hormone administration the aortic tissue proved to contain a lower percentage of chondroitin sulphate A, C anc B.     

Macromolecules in the receptor lymph of campaniform sensilla

Summary The receptor lymph of campaniform sensilla on the halteres of the blowfly, Calliphora vicina, was analyzed histochemically. Acid mucopolysaccharides were demonstrated by a test for iron-binding capacity (Hale-reaction). Further characterization by enzyme treatment showed that the receptor lymph contains hyaluronic acid and/or chondroitin sulfate. Ultrahistochemical studies gave evidence for glycoproteins in the inner and outer receptor lymph space. The significance of acid mucopolysaccharides for arthropod sensilla is discussed.     

Cytochemical localization of acid mucosubstances within intestinal tissue stages of Eimeria acervulina and E. necatrix

Synopsis The distribution of acid mucopolysaccharides in the intestinal tissue stages ofEimeria acervulina andE. necatrix has been investigated. The results show that acid mucopolysaccharides are present in the asexual stages of both species and in the sexual stages ofE. acervulina. It is suggested that an esterified hyaluronic acid-like molecule is present in the cytoplasm of schizonts, merozoites, microgametocytes, macrogametocytes and oocysts as well as in the plastic granules of macrogametocytes and the oocyst wall. Sulphated mucopolysaccharides of the chondroitin type occur as additional components in the cytoplasm of the above stages, as well as in large amounts in the perinuclear area of macrogametocytes and oocysts. The functional significance of acid mucopolysaccharides in the coccidial stages is discussed.     

Morphological and histochemical observations on the intestinal epithelium of Ascardia galli (Nematoda: Ascaridida).

The intestinal epithelium of Ascardia galli has been studied with various cytological and cytochemical techniques. It consists of large epithelial cells resting on a thick collagenous basal lamina. Their luminal surface is provided with microvilli. The intestinal cells store considerable amounts of glycogen and neutral lipids. Some intracellular granular inclusions, which stain for proteins, phospholipids and lipoproteins, are distributed throughout the cytoplasm. The brush border is composed of microvilli whereas the outer surface coat consists of saliva resistant PAS-positive material. The detailed histochemical analysis of surface material has revealed that it is composed of nonacetylated acid mucopolysaccharides rich in hyaluronic acid with carboxylate polyanions. The brush border shows intense activities of acid phosphatase and glucose-6-phosphatase, moderate of ATPase, and lipase, weak of 5'-nucleotidase. Acid phosphatase-positive intracellular structures are seen in the intestinal epithelium which form distinct aggregations.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: hyaluronic acid, heparitin sulfate, and keratan sulfate

Circular dichroism spectroscopy has been used to study the interactions of hyaluronic acid, heparitin sulfate, and keratan sulfate with cationic polypeptides. The results indicate that the presence of these mucopolysaccharides has an effect in the conformation of poly(L -lysine) and poly(L -arginine), such that the former adopts the “random” form and the latter takes up the α-helical conformation, rather than the “charged coil” form expected at neutral pH. The relative strengths of the interactions can be judged from the melting temperatures above which they are disrupted. Both the stoichiometry and the strength of the interactions depend on the position, number, and type of anionic groups attached to the polysaccharide backbone. Such considerations place the six common mucopolysaccharides in order of increasing strength of interaction: hyaluronic acid < chondroitin 4-sulfate < heparitin sulfate < chondroitin 6-sulfate < keratan sulfate ? dermatan sulfate. These differences should be paralleled by differences in the interaction of the mucopolysaccharides with collagen and fibrous proteins.     

Selective exposure of mucopolysaccharides is involved in macrophage physiology.

Surface and intracellular mucopolysaccharides of guinea-pig peritoneal macrophages maintained in suspension and monolayer culture were studied. At least five classes of compound (hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate and chondroitin 6-sulfate) were resolved and characterized by electrophoresis and enzymatic degradation. The results reported here suggest that modulation of mucopolysaccharide exposure is involved in macrophage physiology. The possible biological role of surface mucopolysaccharides in macrophage activity is discussed.     

Simple micro method for identification of heparin and other acidic mucopolysaccharides from mammalian tissues

A combination of gel electrophoresis and paper electrophoresis is used for the identification of mucopolysaccharides after the action of specific mucopolysaccharidases. Small amounts of heparin, heparitin sulfates, chondroitin sulfates, and hyaluronic acid can be distinguished in a mixture by this procedure.     

MUCOPOLYSACCHARIDES PRODUCED IN TISSUE CULTURE

1. A method of mass tissue culture has been devised by which, in a relatively short period of time, samples large enough for chemical isolation of mucopolysaccharides can be obtained. 2. Chemical isolation of acid mucopolysaccharides from mass cultures of human fetal skin, human fetal bone, bovine fetal skin, and rat subcutaneous tissue has been carried out. It has been found that the fibroblasts of each of these tissues produce in tissue culture more than one mucopolysaccharide, namely, hyaluronic acid, and a chondroitin sulfate. 3. The chondroitin sulfate produced by fibroblasts of the above tissues in tissue culture was not fully sulfated. The possible significance of this finding is discussed.     

The gallbladder of the Capivara Hydrochoerus hydrochoeris (Mammalia: Rodentia)

The authors studied morphological and histochemically the mucopolysaccharides and proteins in the gallbladder tubular glands and epithelial cells of the capivara Hydrochoerus hydrochoeris. Based on the results the authors concluded: 1. the gallbladder single columnar epithelium consists of secretory, migrating, and goblet cells; 2. in the lamina propria are single coiled tubular glands; 3. goblet and tubular gland cells show neutral and sulphated mucopolysaccharides and sialic acid; 4. columnar cells show neutral mucopolysaccharides and protein radicals; 5. migrating cells show only protein radicals.     

Effects of heparin and other acid mucopolysaccharides on the activity of a cytolytic pore-forming protein (perforin) from cytotoxic T-lymphocytes

A cytolytic protein (perforin) was rapidly purified from a cell line of mouse cytotoxic T-lymphocytes (CTL) by DEAE-cellulose, heparin-Sepharose, and phenyl-Sepharose chromatographies. The purified perforin was activated by heparin, the half maximal concentration being 3-10 ng/ml, depending on the calcium concentration. Other acid mucopolysaccharides, such as chondroitin sulfates A and C, keratan polysulfate, and heparin sulfate, also enhanced the lysis of erythrocytes by perforin, but the concentrations required for activation were more than 100-fold higher than that of heparin. Chondroitin, hyaluronic acid, and keratan sulfate, however, had no effect on the perforin activity. It was suggested that heparin potentiates the lytic activity of perforin and acid mucopolysaccharides may actually be involved in target cell lysis by CTL.     

Histochemical study of polysaccharides in some arteries of the region of the heart of black vulture (Coragyps atratus)

A comparative histochemical study of polysaccharides in some arteries of the region of the heart of black vulture was performed. With some exceptions, no variation in polysaccharides composition in all arteries studied were shown. Arterial walls present a sialo-mucopolysaccharidic complex and it is composed of: sialic acid, neutral mucopolysaccharides, hyaluronic acid and chondroitin sulfates. The concentration of these carbohydrates, mainly of the acidic ones, decrease from the inner portions of the media toward the adventitia.     

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.     

Mucopolysaccharide accumulation in cultured skin fibroblasts derived from patients with mucolipidosis IV.

Increased concentrations of total sulfated mucopolysaccharides (MPS), threefold, and hyaluronic acid (HA), 10-fold, were found in ML IV fibroblast extracts when compared to normal controls. Such accumulations altered the distribution of MPS:HA comprised 70% of total MPS in ML IV but only 30% in control cells. Intracellular sulfated MPS was observed accumulating almost linearly in ML IV fibroblasts. "Pulse-chase" experiments indicate that both HA and the sulfated MPS remain in the ML IV cells for long periods of time; in control cells, they are rapidly removed as low molecular weight, dialyzable fragments. These data suggest that the MPS accumulation in ML IV fibroblasts, is the consequence of a catabolic block, probably involving the lysosome.