Mucopolysaccharide Production Research Articles

Control over acid mucopolysaccharide production of cells in vitro

• Serum addition to stationary chick embryo fibroblasts in vitro causes a 300% increase in acid mucopolysaccharide production, this increase being linear both with time and concentration of serum added. At the same time, serum-stimulated production is reduced in cells which are actively dividing. The increase in acid mucopolysaccharide production caused by serum is in some manner dependent upon RNA synthesis, since the addition of actinomycin D reduces its effect by 50% within 4 h. A correlation between acid mucopolysaccharide production and chondroitin sulphotransferase activity (EC 2.8 2.5, 3'-phosphoadenyl 5'-phosphosulphate: chondroitin sulphotransferase) is seen. Both increase to over 400% of control values 24 h after serum addition, and both are sensitive to the presence of actinomycin D. • Serum control over production of these extracellular molecules is demonstrated and may be linked to the mitotic activity of the cell. It is proposed that this control by serum over acid mucopolysaccharide production can provide one mechanism for the cell to alter its external environment and thus affect events such as cell to cell interactions and hormonal signals necessary to cellular functions.

Clinical, electron-microscopic, and histochemical investigations of conjunctivitis lignosa (author's transl)

A case is briefly described in which a typical conjunctivitis lignosa appeared after the eye had suffered lime burns. In order to help clarify the morphological connection between mucopolysaccharide production and fiber development in the tumor tissue which occurred after the burn, samples were examined histologically, histochemically and with the use of the electron microscope. The tumor had a cartilage like consistency. Its structure could be devided into three regions. Region A is the pseudo-membrane. It has root like extensions which anchor it to the underlying tissue, and which morphologically appear partially homogeneous and partially fibrous. Blood cells and cell remnants are included in the tissue of the pseudomembrane. The histochemical examination of the pseudomembrane did not present a uniform picture. Along with small amounts of dermatan-sulfate and chondroitin-sulfate B the membrane probably contained a rather large amount of hyaluronic acid. The pseudomembrane borders on a granular tissue (Region B) which is distinguished by the wide metachromatic sheathes of the blood vessels found in it and the particularly large number of active fibroblasts along its edges. The silver impregnation method and the electron-microscopic examination showed that the vascular sheathes consist of bundles of reticular fibers which constitute a three-dimensional network. A similar sort of sheath was observed around the fibroblasts. Chondroitin-sulfate makes up the largest fraction of the mucopoly-saccharides near the fibers and appears particularly concentrated at the intersections of the fibers, although it is also diffusely distributed as well. Dermatan-sulfate (or heparan-sulfate) is found only in the mucopolysaccharide sheath of the fibers themselves. The deep region of the tumor (Region C) consists almost exclusively of blood vessels, their sprouts and the fibroblasts which, with their wide fibrous sheathes, almost fill the spaces between the blood vessels. The reticular fibers and their mucopolysaccharide sheathes have the same structure as that observed in Region B, The fact that the mucopolysaccharides did not appear in plaques but rather as bound primarily to the fibers is grounds for suggesting that a fiber development disorder, probably stemming from the pericytes and fibroblasts rich in ergastoplasm and fibrilles, could play the principle role in conjunctivitis lignosa. The cartilagen like consistency of the tumor could be a result of the arrangement of the fibers and their mucopolysaccharide sheathes. Brief remarks are included concerning the therapeutic consequences of the study.

The effect of lex on UV sensitivity, filament formation and lambda induction in ion mutants of Escherichia coli.

The lex mutation was transduced into strains K12 lon and B (lon). Survival after UV was identical with that of isogenic exrA strains. lex like exrA suppressed UV induced filament formation without affecting the mucopolysaccharide production of K12 lon strains. lex mutants were found to suppress spontaneous and UV induction of λ prophage. The kinetics of suppression were similar to those described for the exrA (λ) mutant. The conclusion is that lex and exrA probably represent mutations within the same cistron.

Rous sarcoma virus-induced tumors in marmosets: characteristics of tumor cells and host cell-virus interrelationships.

Thirty-eight tumors were induced in white-lipped marmosets(Saguinus nigricollis, S. fuscicollis) by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV). They were analyzed for viral genome, virus-expressed antigen(s), chromosomal aberrations, and the production of acid mucopolysaccharides (AMPS). Tumors established in cell cultures had morphologic characteristics typical for Roustumor cells of fowl and other mammalian species. The viral genome was demonstrated in 7 of 18 tumors by cocultivation with live, X-irradiated, or disrupted chicken cells. No group-specific antigen was detected by complement fixation tests (COFAL) in tumors examined, and only a few animals with a long latent period developed COFAL antibodies. Although no specific chromosomal aberrations accompanied marmoset SR-RSV-induced tumors, chromosomes were often broken and rearranged, which wasattributed to inadequate cell nutrition in fast-growing and poorly vascularized tumors. Large quantities of AMPS were demonstrated in some tumors, and all tumors had some AMPS on the periphery of tumors infiltrating surrounding muscles. Infectious viruses isolated from marmoset SR-RSV-induced tumors had envelope antigens similar to either subgroup A or D of avian sarcoma-leukosis viruses, as did the original SR-RSV used for marmoset inoculation.

Collagen and mucopolysaccharide production in growing lung fibroblasts exposed to chrysotile asbestos

Growing lung fibrolasts exposed to Rhodesian chrysotile asbestos for 24 days barin vitro show a pronounced fibrogenic response which is reflected by an increase in the level of cell mat collagen and an alteration in the hyaluronic acid/chondroitin sulphate ratio in the culture medium. The pattern of cell growth protein formation and collagen production throughout the culture period is considerably different in normal and chrysotile-treated cultures. A ‘control dust’, glass fibre, has ony a slight initial fibrogenic effect which is no longer evident at the termination of the culture period.

Production of mucopolysaccharides by normal and transformed cells.

The rate of hyaluronic acid and sulfated mucopolysaccharide production was measured for hamster embryo fibroblasts and for general oncogenic lines derived by virus transformation. A striking increase in both the rate of hyaluronic acid synthesis and the amount of cell-associated polymer was observed after transformation by herpes simplex type-2 or SV40 virus. Although no corresponding change was observed for the sulfated polysaccharides, the proportion of heparan sulfate increased significantly after transformation.

Chondroblastoma--the nature of the basic cell. A study by means of histochemistry, tissue culture, electron microscopy, and autoradiography.

The histogenesis of chondroblastoma is controversial; both cartilage cells and reticulohistiocytes have been proposed as the cell of origin. We have examined a humeral chondroblastoma histochemically and autoradiographically for mucopolysaccharide production and compared the tumor cells ultrastructurally with normal fetal and adult cartilage cells. The chondroblastoma cells produced sulfated acid mucopolysaccharides and ultrastructurally showed features of both fetal and adult cartilage cells. The relationship of the chondroblastoma cells to reticulohistiocytes has been examined, and we have concluded that origin of the tumor from reticulohistiocytes is unlikely and that the chondroblastoma is truly cartilaginous in origin. Since the term chondroblast is used so variably, we have attempted to define this cell functionally and morphologically. The implication that a chondroblastoma is made up of embryonic cells is questionable.

Histochemical demonstration of acid mucopolysaccharide production by tissue cultured epithelial-like cells.

The production of mucopolysaccharides by human epithelial-like cells in vitro has been studied using histochemical techniques. Cells of the human amnion F/L line were grown for 12–14 days in Leighton tubes containing cover slips which were fixed and stained by the Hale, PAS, toluidine blue and alcian blue stains and techniques. Control slides were treated with active and heat inactivated solutions of testicular and bacterial hyaluronidase and chondroitinase prior to staining. Results indicated that material stainable by all the techniques employed was elaborated by the amnion cells and this was partially removable by various combinations of polysaccharases. It is concluded that epithelial-like cells can synthesize and elaborate the ground substance components, hyaluronic acid, chondroitin sulphates and unidentified polysaccharide complexes, in much the same manner as mesenchymal cells and thus contribute to the maintenance of epithelial integrity by de novo production of intercellular substances.

Production of sulfated mucopolysaccharides by established cell lines of fibroblastic and nonfibroblastic origin

Production of sulfated mucopolysaccharides by established cell lines of fibroblastic and nonfibroblastic origin

Initiation of a differentiated function (hyaluronic acid synthesis) by hybrid formation in culture

Gene expression in mammalian cells was investigated by the use of somatic cell hybridization technique in culture. 1. 1. One clonal line, B-6, among hybrids derived from the fusion of mouse and Chinese hamster cell lines was demonstrated to synthesize and secrete hyaluronic acid into medium. 2. 2. Although B-6 line comprised floating, round and single cells, they had a highly differentiated function, characteristic of only a few fibroblastic lines in culture. 3. 3. This property was barely present in either both the parental or the other three hybrid lines obtained, suggesting that the hybridization event initiated the expression of genes responsible for hyaluronic acid synthesis in the B-6 line alone. 4. 4. A kinetic study of the mucopolysaccharide production revealed that the function of the B-6 line in question operated only during the actively growing phase of the cells, stopping completely in the stationary phase. This suggests the existence of an intimate coordination between active cell growth and hyaluronic acid synthesis. 5. 5. Treatment of B-6 cells with either actinomycin D or cycloheximide which are potent inhibitors of RNA and protein synthesis, respectively, inhibited cell growth and hyaluronic acid synthesis to the same degree, whereas in the presence of hydroxyurea, an inhibitor of DNA synthesis, hyaluronic acid continued to be produced by the cells in spite of the absence of multiplication.

Morphogenesis and functional differentiation of the rat parotid gland in vivo and in vitro

ABSTRACT In comparison with the submandibular and the sublingual glands, the parotid develops slowly in the rat. The foetal rudiment appears a day later than that of the submandibular gland and the formation of adenomeres is slower, leading to a more diffusely branched structure. Cytodifferentiation, in the form of traces of mucopolysaccharide in the tubules and terminal buds, begins at, or just before, birth. There is a transitory increase in mucopolysaccharide production for a few days after birth until the presumptive acinar cells become pyramidal in shape with basal nucleus and granular cytoplasm. Amylase activity of the gland begins to rise between the second and third day after birth and reaches the adult level at weaning. That of the submandibular gland remains at the foetal level. Parotid rudiments were cultivated on a film of agar over a medium of fowl plasma and chick embryo extract. The oxygen in the gas phase of air and 5 % CO2 was increased to 50 % after the first 9 days in vitro. Under these conditions the mass of the rudiments increased tenfold during 18 days cultivation and the initially unbranched rudiment formed adenomeres in which the cytodifferentiation followed the same course as in vivo. The rise in amylase activity of the explants was only slightly delayed compared with that in vivo, suggesting that systemic or environmental factors are not obligatory in the early postnatal development of the rat parotid.

Aortic media explants cell proliferation and production of mucopolysaccharides, collagen, and elastic tissue

Segments of pig aortic medial tissue, cultured in a semisynthetic medium, develop a new growth starting about the fourth day. In the first week most cells in this new growth are primitive cells and fibroblast-like cells with only a few modified smooth muscle cells. At this period, only mucopolysaccharides can be identified in the extracellular substances. By the third week, the great majority of cells are modified smooth muscle cells. The extracellular substance is composed of collagen and elastic tissue in addition to mucopolysaccharides. Since the two phenomena, cell proliferation and the production of these specific extracellular substances, are among the most prominent features of early atherosclerosis, it would seem that this in vitro system could be used advantageously in experiments designed to study the role of individual factors implicated in atherogenesis.

Influence of fibroblastic collagen and mucopolysaccharides on Hela cell colonial morphology

Human fibroblasts in vitro (a) synthesize collagen and acid mucopolysaccharides and (b) when grown together with HeLa cells, induce a change in the colonial morphology of the latter. This study demonstrates that the induction of colonial changes by the fibroblasts is not necessarily causally related to the production of collagen and acid mucopolysaccharides.

Activity of aminopeptidases (amino naphthylamidases) during early limb bud differentiation in the chick embryo.

AbstractThe morphologic changes in the differentiation of chick embryo limb buds from Hamburger‐Hamilton stages 19 through 34 were correlated with biochemical measurements of hydroxyproline biosynthesis and aminopeptidase enzyme activity. The distribution and quantitative approximations of collagen, mucopolysaccharides and aminopeptidase activity were observed by histochemical methods. Aminopeptidase enzyme activity increased after four days of incubation (stages 24‐25) concomitantly with the formation of precartilage masses (development of lakes of intercellular mucopolysaccharides). Histochemical preparations revealed the mesodermal cells in the precartilage masses to have an increased aminopeptidase enzyme activity. It was also noted that the enzyme activity in the ectodermal ridge was in excess of that found in the subjacent mesodermal cells or adjacent epithelium. Wet weights of limb buds revealed a constant rapid rate of growth throughout the period of study. However, aminopeptidase enzyme activity was not biochemically detectable until the onset of increased mucopolysaccharide synthesis, when cartilaginous differentiation begins. At five days (stage 26), hydroxyproline was first detectable biochemically and collagen histochemically. This was concomitant with the onset of hydroxyproline production in the embryo in toto, reported previously. The curve of acceleration of hydroxyproline production per milligram of bud mesoderm was less than the acceleration of aminopeptidase activity. The aminopeptidase enzyme activity in the premuscle masses and ectodermal‐mesodermal interface, where histochemically detectable collagen was located, was at the same level as the intervening tissues. After stage 19, limb bud mesoderm was cultured in vitro for seven days. Hydroxyproline production was not biochemically detectable and collagen not stainable, though mucopolysaccharide production was extensive. No muscle differentiation occurred and cell proliferation was minimal. Aminopeptidase enzyme activity had increased 20‐fold. It is suggested that at least part of the aminopeptidase enzyme activity of limb buds is associated with mucopolysaccharide synthesis. There was no evidence that it was associated with mesodermal cell proliferation, muscle differentiation, or hydroxyproline production.

Vitamin C deficiency and connective tissue. V. Fractionation of acid mucopolysaccharides in carrageenan-granulomas.

SummaryThe influence of ascorbic acid deficiency on the production of acid mucopolysaccharides was studied on carrageenan-induced granulomas 5,7,10 and 14 days old. By column chromatography, using ECTEOLA cellulose, three fractions of acid mucopolysaccharides were separated, viz., a hyaluronic acid-like and a chondroitin sulfate-like fraction and besides, a residual fraction. By means of infrared spectrophotometry of 14-day-old scorbutic granulomas, absorption bands were revealed representing group frequencies characteristic of acid mucopolysaccharides. The early biochemical alterations in scorbutic granulomas were characterized by an increase in the concentration of acid mucopolysacharides, which was caused by higher concentrations both of a hyaluronic acid-like fraction and a chondroitin sulfate-like fraction.

Vitamin C deficiency and connective tissue. IV. Determinations of mucopolysaccharides and collagen in carrageenan-granulomas.

SummaryThe influence of ascorbic acid deficiency on the production of collagen and acid mucopolysaccharides in carrageenan-induced granulomas is studied. Ascorbic acid deficiency results in the formation of a granuloma, which is poor in hydroxyproline, an animo acid specific of the collagen molecule. Furthermore, the acid mucopolysaccharides of the ground substance are changed, both qualitatively and quantitatively. The concentration of hexosamine and uronic acid increases during development of granulomas in ascorbic acid-deficient animals, and in granulomas 10 and 14 days old the concentration is increased significantly as compared with control granulomas. At the same time a decrease in the fucose concentration is observed, which is indicative of a decrease in the glycoproteinbound hexosamine. Hence, the increase in the hexosamine content is presumably caused exclusively by an increase in the content of acid mucopolysaccharides. The carbazole to orcinol ratio falls significantly during the development of normal control granulomas, presumably as a manifestation of a relative increase in the content of chondroitin sulfate B. During ascorbic acid deficiency, acid mucopolysaccharides are formed with a higher carbazole to orcinol ratio than that found in animals adequately supplied with ascorbic acid.

Observations on the ultimobranchial gland in small wild and laboratory mammals, with special reference to the histochemical localization of polysaccharides

AbstractThis is the last of a series of observations involved in a comparative histological study of the ultimobranchial body in vertebrates. Complete serial longitudinal or transverse paraffin sections were made of formol alcohol, Bouin's, and formalin‐fixed ultimobranchial and thyroid tissues that had been removed from adult specimens of the cat, cotton rat, ground squirrel, Guinea pig, kangaroo rat, bushytailed wood rat, rabbit, and raccoon. The alcian blue‐PAS, aldehyde fuchsin‐alcian blue, mucicarmine, alcian blue‐safranin, Best's carmine, and toluidine blue staining procedures show that the glandular mammalian ultimobranchial tissue is capable of producing considerable amounts of various mucosubstances, particularly the acid mucopolysaccharides, and other carbohydrate‐containing materials. The carbohydrate moiety has been histochemically identified as mucopolysaccharides possessing acidic groups, those having both carboxyl and ester sulfate groups; and as glycogen, muco‐and glycoproteins and other carbohydrate‐protein complexes. The differences in the intensity and localization of a given staining reaction reflect not only individual and species variations but also significant differences in the position of the ultimobranchial tissue in relation to the thyroid gland. An accessory thyroid lobule found near parathyroid IV and thymus IV in a bushy‐tailed wood rat may represent a cystic manifestation of the ultimobranchial body because its follicles varied greatly in size and were lined by a thick stratified squamous epithelium. The possible hormonal mechanisms (TSH, thyroxine) involved in the regulation of ultimobranchial activity are discussed in relation to the gland's production, storage, and release of mucopolysaccharides and other secretory substances.

Human dermal fibroblasts in monolayer tissue culture. Effect of ascorbic acid analogues.

Abstract The effect of some ascorbic acid analogues and two other reducing substances on collagen synthesis, as well as on cell growth and acid mucopolysaccharide production, was studied in monolayer tissue culture of human dermal fibroblasts. d-isoascorbic acid was as effective as l-ascorbic acid in promoting collagen synthesis if the comparison was made at relatively high concentrations such as 200 μg per milliliter of culture medium. Dehydroascorbic acid was ineffective when it was added alone, but was effective when added with cysteine, although inferior to equivalent amounts of ascorbic acid. Neither cysteine nor hydroquinone affected hydroxyproline synthesis. Cysteine seemed to have a sparing effect when it was added with ascorbic acid. It is suggested that the active site of ascorbic acid in hydroxyproline synthesis is its hydroxyradical or radicals and that there is a relative lack of stereospecificity for this vitamin.

THE EFFECT OF ASCORBIC ACID ON HUMAN DERMAL FIBROBLASTS IN MONOLAYER TISSUE CULTURE.

Abstract The effect of ascorbic acid on collagen and acid mucopolysaccharide synthesis and on cell growth has been studied with the use of monalayer tissue cultures of fibroblasts obtained at the time of operation. The addition of ascorbic acid in quantities as low as 50 μg per milliliter of a nutrient medium which consists of 83 per cent Eagle's basal medium, 15 per cent heat deactivated human serum, and 2 per cent embryonic extract 50 , and which is supplemented with antibiotics affected the collagen metabolism of the cells dramatically. In this system, human dermal fabroblasts were capable of synthesizing collagen as long as 3 months after explantation, whereas without supplementation of vitamin C no appreciable collagen synthesis could be observed. Ascorbic acid additions generally enhanced cell growth, whereas they did not show a consistent effect on acid mucopolysaccharide production. Hyaluron c acid rather than sulfated acid mucopolysaccharides ivas synthesized in all the groups whether or not ascorbic acid was added. Possible mechanisms of collagen synthesis are discussed.

Mucopolysaccharide Production By Fibroblasts Derived from Human Buffy-Coat Cultures in vitro

Grossfeld(l) has shown that fibroblasts from different tissues cultivated in vitro are able to produce acid mucopolysaccharides (AMPS) and release them into the medium. AMPS belong to the group of hyaluronic acid and chondroitin sulphates A and C, are highly polymerized, and give positive mucin clot and hyaluronidase tests(2).Since fibroblast-like cells are known to originate in cultures of buffy-coat and hemapoie-tic tissues(3,4), it seems of interest to study the AMPS production in human white blood cell cultures undergoing fibroblastic transformation.Materials and methods. Peripheral human blood buffycoat was obtained by venipuncture and centrifugation at 600 × g with 0.1 mg/ml of heparin (Liquemine Roche) for each 5 ml of blood and phytoagglutinine (Difco), 0.25 ml for each 10 ml of blood. The first 1 or 2 ml extracted from the vein was discarded to avoid contamination with tissue cells. Buffycoats from 10 ml of blood were explanted in Earle's flasks with a layer of isologous plasma and 5 ml of liquid...