Mucopolysaccharide Synthesis Research Articles

Alcohol as a Stimulus to Hepatic Fibrogenesis<xref ref-type="fn" rid="fn1"><sup>*</sup></xref>

Examination of mouse liver using biochemical and autoradiographic techniques has demonstrated a very transient rise in the synthesis of collagen and sulphated mucopolysaccharide in the sinusoidal walls following a single large dose of alcohol. Mice fed a diet with alcohol providing about one-third of the total energy intake often showed microscopic evidence of an increase in hepatic connective tissue. In no case did this progress to established liver cirrhosis. Similar techniques have been used to study human hepatic biopsy material in vitro. In chronic active liver disease including that occuring in alcoholics there is a notable increase in sulphated mucopolysaccharide synthesis while the collagen proline hydroxylase content of this tissue is also elevated.

Achondroplasia in the embryonic chick: its potentiation by cortisone acetate and alleviation by vitamin C.

Thallium sulfate induced achondroplasia has been studied in the embryonic chick by culturing the tibiae from normal embryos in embryo extract from thallium sulfate treated embryos; by injecting normal embryos with homogenates of thallium sulfate treated embryos; and by the coinjection of thallium sulfate and cortisone acetate or vitamin C, and analysis of the tibiae for growth, histology, histochemistry (collagen, acid mucopolysaccharide, glycogen, calcium, alkaline phosphatase), hexosamine, and hydroxyproline activity.Tibiae from untreated embryos organ cultured in embryo extract from thallium sulfate treated embryos, and tibiae of host embryos injected with homogenates from thallium sulfate treated embryos, exhibited normal growth, histology, histochemistry, hydroxyproline, and hexosamine activity. Thus the achondroplasia observed in thallium sulfate treated embryos does not result from the production of an inhibitor to normal chondrocyte growth and function which is transmissible in saline extracts. This is in contrast to some genetic achondroplasias.Coinjection of thallium and vitamin C or of thallium and cortisone acetate (agents increasing or decreasing collagen and hexosamme-containing acid mucopolysaccharide synthesis respectively) resulted in potentiation of the syndrome in the presence of cortisone acetate, and alleviation of the syndrome in the presence of vitamin C. Vitamin C prevented the production of the defective cartilage matrix characteristic of the long bones of embryos treated with thallium sulfate alone.It was concluded that abnormalities in acid mucopolysaccharide and collagen release from chondrocytes and deposition in the cartilaginous matrix were possible causes of the chondrocyte necrosis, achondroplasia, and micromelia characteristic of the achondroplastic syndrome.

Synthesis of RNA, protein, cellulose, and mucopolysaccharide and changes in the chemical composition of Hartmannella culbertsoni during encystment under axenic conditions.

SYNOPSISHartmannella 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.

Cytochalasin B: lack of effect on mucopolysaccharide synthesis and selective alterations in precursor uptake.

Synthesis and secretion of mucopolysaccharide in mouse 3T3 fibroblasts and in embryonic submandibular glands are unaffected by amounts of cytochalasin B that alter the morphology of these cells and tissues. The drug markedly and reversibly inhibits incorporation of [(3)H]glucosamine into mucopolysaccharide by preventing cellular uptake of the precursor, but does not affect incorporation of radiosulfate. Cytochalasin does not alter DNA, RNA, or protein synthesis, but stimulates the uptake of orotic acid and markedly inhibits the uptake of glucose. These selective effects on the transport of small molecules suggest that the primary action of the drug may be on cell membranes. Since processes unrelated to microfilament disruption may be altered by cytochalasin, great caution must be exercised in interpreting studies with the drug.

The Effect of Dibutyryl Cyclic Adenosine Monophosphate on Synthesis of Sulfated Acid Mucopolysaccharides by Transformed Fibroblasts

Abstract The effect of dibutyryl-cyclic adenosine 3',5'-monophosphate and theophylline on the incorporation of [35S]sulfate into acid mucopolysaccharides by 3T3 and SV3T3 fibroblasts has been studied. SV40 transformation of 3T3 fibroblasts produced a marked decrease in the rate of synthesis of acid mucopolysaccharides, in agreement with the data of others. When the transformed cells were grown in medium supplemented with dibutyryl-cyclic AMP and theophylline for 3 days, they secreted, on the basis of cell protein, approximately 250% more labeled acid mucopolysaccharides during a 24-hour labeling period than did the nontreated controls. The secretion of a variety of sulfated acid mucopolysaccharides was increased including chondroitin-4- and -6-sulfate and dermatan sulfate. Comparison of the rate of sulfate incorporation into acid mucopolysaccharides and the rate of loss of previously labeled acid mucopolysaccharides from the cells indicated that the effect was due to a greater rate of synthesis of these compounds at the time of labeling rather than due to a decrease in the rate of their degradation.

Coenzyme competition and precursor specificity during teratogenesis induced by 6-aminonicotinamide

The teratogenic mechanism of the nicotinamide analog 6-aminonicotinamide (6-AN), is thought to be inhibition of energy exchange reactions which in turn selectively interfere with synthesis of mucopolysaccharides. Nicotinamide and 6-AN were administered singly or in combination for studying micromelia in the embryonic chick limb. These coenzyme competition, protection, and reversibility experiments suggest that cells of 4-day embryos readily accept either nicotinamide or 6-AN. Preloading with nicotinamide 1–24 hours prior to exposure to 6-AN prevented teratogenicity of 6-AN. An excess of nicotinamide up to 2 times the maximal protective dose given 2 hours or more after 6-AN did not offset the teratogenicity of 6-AN. Once exposed either to 6-AN or to nicotinamide the cells do not readily accept either alternate molecule. This mutual exclusivity enabled demonstration that 6-AN is 80% effective as a teratogen in 4-day chick embryos within 2 hours after its administration. Duration of the effectiveness of a standard dose of 6-AN given to a 4-day embryo was studied by a 30-minute terminal label with 35S-sulfate. Maximum inhibition was seen 3 days after treatment and recovery to control values occurred in about 4 1 2 days. Because limb teratogenicity is a result of atypical chondrogenesis and because the experiments above were performed on mixed tissues of whole embryos or whole limbs, early rudiments of tibia at 9, 10, and 11 days were isolated and tested with 6-AN and labeled precursors. It was found that 2 hours of exposure of bone rudiments to 6-AN was sufficient to demonstrate selective inhibition of 35S incorporation while amino acid incorporation remained similar to controls. The effect of 6-AN in the limb of the chick embryo is prompt, demonstrable in 2 hours and temporarily irreversible. Recovery is seen in 4 to 5 days, but distortion of the cartilage model results in micromelia.

The effects of the nicotinamide sensitive teratogen 3-acetylpyridine on chick limb mesodermal cells in culture: biochemical parameters.

Abstract3‐acetylpyridine (3AP) causes severe muscular hypoplasia in vivo and seems to potentiate chondrogenic expression of limb bud cells in vitro. In both cases nicotinamide completely relieves these effects. In cell cultures, 3AP causes a substantial drop in nicotinamide incorporation. This decreased incorporation represents not only decreased cellular pools of nicotinamide but also decreased DPN‐TPN pools. 3AP also decreases the rate of RNA, protein, and mucopolysaccharide synthesis. 3AP does not affect the rate of DNA synthesis which is constantly decreasing as chondrogenic expression increases. Even though the rates of macromolecular synthesis seem to be reduced, the specific activity of an obligatory enzyme for mucopolysaccharide assembly, UDP glucose pyrophosphorylase, dramatically increases with 3AP‐treatment. This is interpreted to mean that 3AP potentiates of cartilage synthesizing machinery which is observed as a potentiation of chondrogenic expression. These data are discussed with regard to nicotinamide's proposed role in affecting the development of chick limb mesodermal cells into muscle and cartilage producing phenotypes. It is proposed that alterations in local concentrations of essential molecules like nicotinamide, DPN, or TPN can control phenotypic expression.

Synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and platelets of the rat, an anlysis by electron-microscope autoradiography.

ABSTRACT Electron-microscope autoradiography has been used to investigate the synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and blood platelets. Following 10-min incubation of bone marrow with 35S-sulphate in vitro the majority of the activity in megakaryocytes was associated with the Golgi apparatus, but a substantial proportion was associated with other cytoplasmic organelles, suggesting either rapid transport or sulphation of mucopolysaccharide outside the Golgi apparatus. Three hours after the intravenous injection of 35SO4 only a small proportion of the total activity was associated with the Golgi apparatus, most being associated with demarcation membranes and dense granules, while 12 h after injection almost all the activity was associated with demarcation membranes and granules. A rising proportion of activity localized solely on the demarcation membranes suggested that they may possess some activity of their own. Autoradiographs of blood platelets prepared 72 h after the injection of 35SO4 were analysed. It was shown that most of the activity was associated with the a-granules, but there was strong evidence that the platelet membrane possessed a low level of activity.

In vitro synthesis of acid mucopolysaccharides by human gingival fibroblasts.

In vitro synthesis of acid mucopolysaccharides by human gingival fibroblasts.

Inhibition of collagen cross-linking associated with β-aminopropionitrile-induced cleft palate in the rat

Lathyrogenic compounds have been shown previously to induce cleft palate in the rat when administered during gestation. The present study examines the induction of cleft palate by the lathyrogen, β-aminopropionitrile (BAPN) and attempts to correlate this with the effect BAPN has on the cross-linking of embryonic collagen. It was found that 98% cleft palate fetuses could be produced by a single oral dose of 600 mg/kg BAPN only when administered during a 24-hr period of gestation beginning with day 15, whereas cleft palate was not induced on day 14 or 16. BAPN did not affect the synthesis of sulfated mucopolysaccharides or proteins, including collagen, in the embryo, but a dramatic and reversible inhibition was observed in collagen cross-linking. Maximum inhibition occurred 24 hr after BAPN administration, whereas no effect was seen after 48 hr. A correlation was observed between the inhibition of collagen cross-linking and cleft palate formation when BAPN was given in various concentrations as a single oral dose on day 15. It is suggested that the inhibition of collagen cross-linking in the embryo may play a role in cleft palate induction by BAPN.

Subcellular sites for synthesis of glycosaminoglycans (mucopolysaccharides) of rabbit bone marrow cells

The enzyme activities involved in the incorporation of glucuronic acid into endogenous chondroitin sulfate and the incorporation of N-acetyl galactosamine into exogenous oligosaccharide were assayed in various subcellular fractions obtained from rabbit bone marrow cells consisting to more than 90% of myeloid cells. Both the granule and the microsomal fraction contained these enzyme activities. Among the microsomal subfractions (light smooth endoplasmic reticulum, heavy smooth endoplasmic reticulum and rough endoplasmic reticulum) the highest enzyme activities were present in the light smooth endoplasmic reticulum. Isopycnic centrifugation in sucrose density gradients showed that most of the microsomal N-acetylgalactosamine transferase activity banded at a density of approx 1.17 g/ml while the granule enzyme banded at a density of 1.24 g/ml. These findings are consistent with the theory of synthesis of mucopolysaccharides on microsomal or Golgi membranes for subsequent incorporation into cytoplasmic granules produced in the Golgi apparatus of the granulocyte.

Metabolic activity in the saccus vasculosus of the rainbow trout, Salmo gairdneri (Richardson).

Labelling experiments to test mucopolysaccharide synthesis in the saccus vasculosus of the rainbow trout gave negative results and provoked analysis of succinic dehydrogenase activity in the coronet cells. Such activity was almost absent in the fresh-water living rainbow trout but was intense in sea water adapted individuals as well as in a sea water teleost, the flounder. Conversely, in sea water adapted rainbow trout which had been transferred back to fresh-water, activity had again decreased. In the sea water adapted rainbow trout high succinic dehydrogenase activity was paralleled by strongly positive staining with alcian blue, indicating the presence of mucopolysaccharides. The latter fishes also showed incorporation of N-acetyl-3H-glucosamine. Our findings indicate a role for saccus vasculosus in adaptation for osmoregulation in sea water and favour the hypothesis of a secretory function for the coronet cells.

Differentiation of cartilage and bone from common germinal cells. II. Inhibition of chondrogenesis induced by -aminoproprionitrile.

Injection into chick embryos incubated for nine days, of β-aminopropionitrile (BAPN) at doses above 0.312 mg/embryo prevented the formation of the cartilage normally present on the membrane bones from the eleventh day of incubation onwards. The histogenesis of this inhibition is described. BAPN increased the proportion of soluble collagen without altering total collagen synthesis and decreased the synthesis of hexosamine-containing acid mucopolysaccharides within the membrane bones. The relationship between cross-linking of collagen, collagen solubility, synthesis of acid mucopolysaccharides and mechanical stimulation (the extrinsic factor initiating chondrogenesis in this system) in the formation of cartilage from bipotential germinal cells on the membrane bones is discussed.

Theories of the Pathogenesis of Cystic Fibrosis

The basic defect in cystic fibrosis is not known, but current evidence points to action of a humoral substance on cells of the exocrine glands. The resulting interaction increases metabolic activity within the cell, altering cell membrane permeability, changing electrolyte concentrations, and increasing mucopolysaccharide synthesis.

Functional and structural changes in Drosophila salivary gland cells triggered by ecdysterone

In normal development a complex pattern of changes in the submicroscopic structure of Drosophila hydei salivary gland cells occurs in relation to the onset of secretory activity of these cells prior to puparium formation. The major changes involved in this structural reorganization of cytoplasmic components are as follows. The mucopolysaccharide secretory product present in the form of globules which are randomly distributed over the cytoplasm in the course of the third instar migrate to, and accumulate at, the cell apex. The arrangement and number of microvilli undergo drastic changes involving elongation and increase in number of the villi. The RER, which is originally distributed randomly, is reorganized into whorls and vesicles. These major changes can be experimentally reproduced by administration of the steroid moulting hormone derivative ecdysterone to larvae in the middle of the third instar. Administration of ecdysterone to early third instar larvae, in which salivary glands are still inactive with respect to mucopolysaccharide synthesis, results in only a partial reproduction of the features displayed by middle third instar glands. The changes in the arrangement and number of microvilli do not occur in the early third instar glands. The changes observed in the early third instar salivary gland cells indicate that the onset of mucopolysaccharide synthesis is inhibited by the steroid treatment. Support for this assumption is provided by studies on the features of the RER and Golgi systems in middle third instar salivary gland cells. On the basis of this investigation it may be suggested that the steroid ecdysterone is an effective trigger of mechanisms active in cytodifferentiation. It seems that not only secretion of mucopolysaccharides is initiated by the steroid, but also the synthesis of this substance inhibited.

The glucuronidation pathway

Salicylic acid (SA) and acetylsalicylic acid (ASA), unlike cortisone, promote the release of lysosomal enzymes rather than protecting rat liver lysosomal membrane. Salicylic acid inhibits the oxidation of uridine-5-diphosphoglucose (UDPG) competitively with nicotinamide adenosine dinucleotide (NAD) and noncompetitively with UDPG. It also competitively inhibits the transferring of glucuronyl group of uridine-5-phosphoglucuronic acid (UDPGA) to the phenolic acceptor. The wound-healing retardation action of salicylates is probably due mainly to its inhibitory action on mucopolysaccharide synthesis.

Inhibition of mucopolysaccharide synthesis by 5-bromodeoxyuridine in cultures of chick amnion cells

Mucopolysaccharide synthesis has been investigated in cultures of fibroblastic cells prepared from the amnion of 10-day chick embryos. Using glucosamine- 14C as a precursor, the synthesis of hyaluronic acid and chondroitin sulfate has been demonstrated by (i) migration of labeled mucopolysaccharides after electrophoresis in pyridinium formate buffer, (ii) hexosamine content of fractions thus obtained, (iii) susceptibility to testicular hyaluronidase. Material labeled with sulfate- 35S has been identified specifically as chondroitin sulfate A by (i) electrophoresis in zinc acetate, (ii) digestion with chondroitinase AC and separation of products obtained. Cultures at low density incorporate glucosamine almost exclusively into hyaluronic acid, while at high density incorporation into chondroitin sulfate and hyaluronic acid occurs in roughly equal amounts. Growth of amnion cells in the thymidine analog 5-bromodeoxyuridine (BUdR) results in a progressive loss in their ability to synthesize both mucopolysaccharides with little loss in their ability to grow and divide. There is good correlation between the incorporation of BUdR into the cell's DNA and the inhibition of mucopolysaccharide synthesis. Amnion cells suppressed with BUdR are cytologically distinct from functional amnion cells. If BUdR-suppressed cells are allowed to replicate in the absence of the analog they yield a progeny which are cytologically similar to normal cells, and resume active synthesis and secretion of hyaluronic acid and chondroitin sulfate.

Early biochemical changes in the embryonic rat heart after teratogen treatment

AbstractThe effects on mucopolysaccharide synthesis in isolated embryonic rat hearts of in vivo and/or in vitro 6‐aminonicotinamide, dietary‐induced folic acid deficiency, aminopterin, and trypan blue were investigated using uptake of 35S‐sodium sulfate to measure the rate of mucopolysaccharide synthesis. Intraperitoneal injection of 6‐aminonicotinamide on day 11 of gestation caused inhibition of mucopolysaccharide synthesis which was most pronounced at day 13. A similar effect followed in vitro 6‐aminonicotinamide treatment. Maternal folic acid deficiency on days 9–11 of pregnancy caused decreased mucopolysaccharide synthesis in embryonic hearts at day 14. In vitro treatment with the folic acid antagonist aminopterin inhibited the rate of synthesis on days 13–16. In vivo trypan blue on day 9 of pregnancy had no effect on the rate of mucopolysaccharide synthesis. However, in vitro trypan blue treatment of embryonic hearts resulted in a marked depression in synthetic rate. Aminopterin and 6‐aminonicotinamide had no noticeable effect on the rate of protein synthesis in isolated embryonic hearts as determined by measurement of 3H‐leucine incorporation. Thus mucopolysaccharide synthesis in isolated embryonic rat hearts was susceptible to the action of the teratogens used. It is postulated that these teratogens act by blocking a biochemical reaction in the synthesis of mucopolysaccharides and are not affecting the cellular machinery necessary for general protein synthesis.

Acid mucopolysaccharide synthesis by chick amnion cell cultures: Inhibition by 5-Bromodeoxyuridine

Summary Cell lines derived fromthe chick amnion synthesize sulfated and unsulfated acid mucopolysacchardies in vitro. Polysaccharide formation was studied by viscometry of culture medium and by incorporation of labeled glucose and sulfate. Synthesis and deposition of polysaccharides depend upon the age of the cultures during the subculture period. Sparce cultures release most of the AMPS into the fluid phase of the culture medium giving rise to high medium viscosity. As the cultures become dense, more of the AMPS is retained near the cells and forms a metachromatic matrix between cells and medium. In addition, incorporation of labeled glucose into polysaccharide is 10-fold greater in cells from sparse cultures compared with dense cultures. These cyclic alterations in cellular activity are repeated during each subculture period for many months. Incorporation of 5-bromodeoxyuridine into DNa is accompanied by alterations in cell morphology and suppression of polysaccharide synthesis within 1–2 days. These effects can be prevented by simultaneous addition of thymidine. Cells grown in BUdR continue to proliferate and can resume normal activity when removed from the analogue and allowed to divide in untreated medium.

Biosynthesis of UDP N-acetylhexosamine in bovine retina.

In this paper the metabolism of amino sugars in the retina and in the pigment epithelial cells has been studied. Extracts obtained from the retina and from the pigment epithelial cells are capable, in the presence of suitable cofactors, of converting [1-14C]glucosamine into UDP-N-acetylhexosamines. Chromatographic investigations with the use of Dowex resin column have permitted the identification of the principal intermediaries of amino sugar metabolism: GlcNAc, GlcN-6-P, GlcNAc-6-P, UDP-GlcNAc, UDP-GalNAc. Both the retina and the pigment epithelial cells are, further, capable of synthesizing those amounts of acetyl coenzyme A necessary for the formation of acetylated amino sugars. The metabolism of amino sugars in the retina would appear to be much more active than in the pigment epithelial cells; this would indicate that the greater part of the synthesis of mucopoly-saccharides of the interstitial matrix takes place in the retina. In the course of this research, furthermore, the presence of three compounds has been ascertained, the identification of which is still in progress.