The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage.

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, beta-D-xylosides and beta-D-galactosides to the incubation medium. Beta-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, beta-D-xylosides and beta-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.     

Degradation of heparin in mouse mastocytoma tissue

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GE(H)) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GE(H) was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights ( M(w)) of approx. 60000-70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate-protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [(35)S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating (35)S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting (14)C-labelled chondroitin sulphate to the same procedure.     

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.     

Effects of cycloheximide, brefeldin A, suramin, heparin and primaquine on proteoglycan and glycosaminoglycan biosynthesis in human embryonic skin fibroblasts.

(1) We have isolated radiolabelled proteoglycans and glycosaminoglycans produced by human embryonic skin fibroblasts in the presence of (a) cycloheximide to inhibit protein synthesis or (b) brefeldin A to impede transport between the endoplasmic reticulum and the Golgi complex or (c) suramin, heparin or primaquine to interfere with internalization, recycling and degradation. Effects on glycosaminoglycan synthesis were assayed separately by using exogenous p-nitrophenyl beta-D-xylopyranoside (and [3H]galactose) or 125I-labelled p-hydroxyphenyl beta-D-xylopyranoside as initiators. (2) Inhibition of protein synthesis or blocking of transport to the Golgi complex prevented production of most of the proteoglycans with one exception: Cell-associated heparan sulphate-proteoglycan was still produced at 20% of the control level. (3) Treatment with suramin or heparin resulted in decreased deposition of proteoglycan in the pericellular matrix but increased accumulation of cell-associated proteoglycan. Primaquine blocked all proteoglycan synthesis. (4) In the presence of cycloheximide, exogenous beta-D-xyloside initiated galactosaminoglycan production. In contrast, in brefeldin A-treated cells, synthesis was completely abolished. Not even formation of the linkage-region trisaccharide could be detected. (5) These results suggest that exogenous xyloside enters the endoplasmic reticulum and is subsequently transported to the trans-Golgi complex where all further steps involved in glycosaminoglycan assembly takes place. (6) Heparan sulphate proteoglycan produced by brefeldin A-treated cells could be derived from (a) an intracellular pool of preformed core protein located to the trans-Golgi complex, or (b) resident proteoglycan that was either deglycanated/reglycanated or chain-extended. As combined treatment with suramin and brefeldin A markedly reduced cell-associated proteoglycan production, the latter possibility is favoured.     

Effect of cycloheximide on protein biosynthesis in rat liver

1. The liver ribosomes of rats given cycloheximide by intraperitoneal injection incorporate less amino acid into protein than ribosomes from control rat liver when they are incubated in vitro with excess of Sephadex-treated cell sap. The effect is rapid, marked and persistent. 2. Cell sap from liver of cycloheximide-treated animals is inhibitory but the inhibition can be relieved almost entirely by treating the cell sap with Sephadex. No damage has been done to the cell-sap factors: it is suggested that the dissolved cycloheximide in the cell sap causes the inhibition. 3. Cycloheximide added in vitro inhibits amino acid incorporation into protein in the presence or absence of polyuridylic acid. The inhibition is lessened by addition of excess of cell sap but is not abolished. 4. The differences between these results and those obtained with mouse liver (Trakatellis, Montjar & Axelrod, 1965) might arise because of species differences in sensitivity to the drug.     

Effects of cycloheximide on chromatin biosynthesis.

In the presence of sufficient cycloheximide, puromycin or NaCl to quantitatively inhibit protein synthesis in HeLa cells, thymidine incorporation continues at 20% of control rates for 60 to 90 minutes, after which incorporation gradually ceases. Both DNA and protein synthesis revert to control rates in about five minutes after removal of cycloheximide.DNA synthesis in the presence of cycloheximide appears to be a continuation of the replicative process by several criteria. The persistent DNA synthesis in the presence of cycloheximide is abolished by hydroxyurea, which does not inhibit repair synthesis, while ethidium bromide, an inhibitor of mitochondrial DNA synthesis, is without effect. Nuclear DNA is not nicked during incubation in cycloheximide. Low molecular weight Okazaki fragments (4 to 5 S) are both synthesized and processed to high molecular weight DNA in cells treated with cycloheximide. Replication forks, identified in alkaline CsCl gradients by incorporation of bromodeoxyuridine as a density marker just before the addition of cycloheximide, are selectively labeled with radioactive thymidine during DNA synthesis.In the presence of cycloheximide the maturation of DNA intermediates into high molecular weight DNA is defective. All size classes of DNA fragments, normally present during progression of low to high molecular weight DNA, are demonstrable in cells preincubated in cycloheximide for prolonged periods. However, 21 S fragments, intermediate in size between Okazaki pieces and mature, high molecular weight DNA, accumulate in cells treated with cycloheximide, demonstrating a defect in maturation of the 21 S intermediates into high molecular weight DNA. After removal of the cycloheximide, the 21 S DNA fragments are processed to high molecular weight DNA at a significantly impaired rate, requiring about three hours for completion of chain growth as compared to 40 to 60 minutes in controls. The slowed growth of DNA fragments synthesized in the presence of cycloheximide following drug removal is not due to persisting effects of cyeloheximide since DNA synthesis immediately following removal of the drug has chain growth rates similar to that of controls.Pools of chromatin proteins exist in HeLa cells, as demonstrated by a brief, labeled amino acid pulse followed by a chase with cycloheximide. The specific activity of chromatin proteins increases significantly during 60 minutes of cycloheximide inhibition. Histone f2a1 accumulates preferentially during this chase period, suggesting that a supply of this highly conserved histone might be requisite to continued replication.Comparison of chromatin synthesized during cycloheximide treatment with pulse-labeled control chromatin has provided insight into the mechanism of assembly of proteins and DNA into the nucleoprotein complex. The DNA of ch-chromatin² is more susceptible to nuclease digestion than control chromatin, suggesting that it is deficient in protein content. Upon reversal of cycloheximide inhibition, the recovery of nuclease digestibility of ch-chromatin to control values takes two to three hours, a time similar to that required for conversion of the corresponding 21 S chDNA fragments to high molecular weight DNA. Briefly pulse-labeled (30 to 60 s) DNA in control chromatin also has an enhanced susceptibility to nuclease digestion of the same degree as found in ch-ehromatin. The time of recovery of increased nuclease susceptibility of newly made chromatin DNA (via protein addition) to control levels is about 10 to 15 minutes and corresponds to the time required for synthesis of replicon-sized units of DNA.In addition to being nuclease-sensitive, both cycloheximide and newly synthesized (30 to 60 s) chromatin have lighter buoyant densities in CsCl gradients than bulk chromatin. This property exists for only one to two minutes in controls and is probably due to structural properties distinct from those rendering nuclease sensitivity.Limit digests of chromatin by micrococcal nuclease yield a characteristic pattern of polynucleotides when resolved in polyacrylamide gels. The radioactivity profiles of limit digest polynucleotides from control and ch-chromatin are identical, indicating that pre-existing chromatin proteins remain in place on newly replicated DNA in the same fashion as in mature chromatin.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Effect of cycloheximide on nuclear maturation of pig and mouse oocytes

Culture of mouse oocytes in medium with 1 or 100 micrograms cycloheximide/ml did not prevent germinal vesicle breakdown (GVBD). In contrast, GVBD in pig oocytes was absolutely blocked at concentrations of 1, 5, 10, 50 and 100 micrograms cycloheximide/ml, respectively. The inhibition of GVBD was not influenced by the presence or absence of cumulus cells and it was fully reversible. When cycloheximide treatment (5 micrograms/ml) was given after preincubation for 6, 12 and 16 h, GVBD occurred in 15, 46 and 75% of oocytes, respectively. It is concluded that proteins important for GVBD of pig oocytes were present in sufficient amounts at about 12 h of culture. The fusion of pig oocytes in metaphase I to oocytes with an intact germinal vesicle revealed that cycloheximide did not inhibit GVBD induced by maturing ooplasm. Therefore, induction of prematurely condensed chromosomes by the maturing ooplasm did not require protein synthesis. However, continuous protein synthesis was necessary to maintain metaphase I and prematurely condensed chromosomes in a typical configuration.     

Biosynthesis of heparin. Purification of a 110-kDa mouse mastocytoma protein required for both glucosaminyl N-deacetylation and N-sulfation

The polymer modification process in the biosynthesis of heparin/heparan sulfate is initiated by N-deacetylation, followed by N-sulfation, of N-acetylglucosamine units. Chromatography of a detergent extract from mouse mastocytoma on wheat germ agglutinin-Sepharose yielded a protein fraction, eluted with 0.3 M N-acetylglucosamine, that expressed N-deacetylase activity, but only after recombination with proteins that did not bind to the lectin column. In subsequent purification of the active lectin-bound component, all assays were performed following addition of the unbound protein fraction. After two additional chromatography steps, on blue Sepharose and 3',5'-ADP-agarose, the lectin-binding N-deacetylase component had been purified about 4300-fold with an 11% yield and showed essentially a single band, corresponding to an apparent molecular weight of approximately 110,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the purified 110-kDa protein showed that it contained, in addition to the N-deacetylase, N-sulfotransferase activity; however, the expression of N-sulfotransferase activity was independent of additional proteins. Backtracking the N-sulfotransferase through the purification scheme previously applied to the N-deacetylase showed the two enzyme activities to the N-deacetylase showed the two enzyme activities to be cofractionated in each separation step. It is proposed that the expression of glucosaminyl N-deacetylase activity depends on the concerted action of (at least) two protein components, one of which also possesses glucosaminyl N-sulfotransferase activity.     

Effect of puromycin and cycloheximide on glycosphingolipid biosynthesis by PHA stimulated human lymphocytes

The effect of puromycin and cycloheximide on the biosynthesis of neutral glycosphingolipids and gangliosides by PHA stimulated lymphocytes is studied. Under conditions of permanent inhibition of protein synthesis, and depending on the time of lymphocyte stimulation, inhibition of the biosynthesis of neutral glycosphingolipids was either restricted only to certain species or was very low for all glycosphingolipid species. The degree of inhibition of the various ganglioside species was affected by the time of incubation. After transient inhibition of protein synthesis and at times when protein biosynthesis had recovered, neutral glycosphingolipid and ganglioside biosynthesis inhibition was very prominent and did not recover. The possibility is discussed that glycosphingolipid biosynthesis does not depend directly on concurrent nascent peptide formation and it is proposed that inhibition of glycosphingolipid biosynthesis is related primarily to impairment of the endoplasmic reticulum and to inhibition of galactose transferases, secondary to the binding of the inhibitors.     

Proteoglycans of soluble fraction of mouse mastocytoma.

Proteoglycans have been isolated from a high speed supernatant fraction of a mouse mastocytoma by procedures which should minimize alteration of the native protein-polysaccharide molecule. The methods used include in vivo labeling proteoglycans with 35S-sulfate, 3H-leucine and 3H-lysine, centrifugation of the tumor homogenate at 105,000 g, cetylpyridinium fractionation of the supernatant, and further purification of some of the fractions obtained by DEAE-cellulose column chromatography, gel filtration on Sepharose 4B and cellulose acetate electrophoresis. Two major sulfated proteoglycans were obtained, one containing keratan sulfate-like material (KSP-S), the other a heparin-like polymer (HP-S). The presence in HP-S of a compound similar to heparin was confirmed by its digestibility with flavobacterium heparinase. HP-S contained about 4 per cent protein. Glycine was the predominant amino acid, and serine did not appear to be involved in the peptide-carbohydrate linkage. The proteoglycan present in HP-S appeared to be homogeneous when examined using cellulose acetate electrophoresis. KSP-S was found to contain sialic acid and its protein content was significantly higher than that of HP-S. Glutamic and aspartic acids were the most abundant amino acids in KSP-S.     

Effect of retinoic acid on the proliferation and tumorigenicity of mouse mastocytoma cells

Retinoic acid (RA) inhibited the in vitro growth of the mouse mast cell tumor line P₈₁₅ in a dose- and time-dependent manner. The inhibition was accompanied by an increase in the amount of neutral intracellular mucopolysaccharides. Study of cell cycle kinetics showed that exposure to retinoic acid led to a slowing-down of the cell-cycle progression possibly related to a more differentiated cell population disclosed by microscopy with a lower proliferative capacity. In vivo, delays in both tumor appearance and mouse mortality were observed after injecting RA into mice bearing mastocytomas. These results suggest that RA could be of interest in the treatment of human malignant systemic mastocytosis with proliferation of immature mast cells.     

The effect of beta-D-xylosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage in the absence of protein synthesis inhibitors.

Incorporation of [35S]]sulphate, [3H]glucose and [3H]serine into glycosaminoglycans and proteoglycans of embryonic-chicken sternum was measured in vitro in incubation medium containing 4-methylumbelliferyl beta-D-xyloside or p-nitrophenyl beta-D-xyloside at low concentrations, and in the absence of inhibitors of protein synthesis. Incorporation of sulphate was decreased by 80% in incubations in which 1mM-4-methylumbelliferyl beta-xyloside or 2.5 mM-p-nitrophenyl beta-xyloside was present; under these conditions, serum factors stimulated incorporation to only a small extent. When the concentration of the xyloside was decreased tenfold, incorporation of sulphate was inhibited by 60-70%, but when normal human serum or L-3,3',5-tri-iodothyronine or both were also added to the incubation medium, incorporation was markedly stimulated. Experiments in which [35S]sulphate and [3H]glucose were incorporated simultaneously, and enzymic analysis of glycosaminoglycans formed in such experiments, indicated that chondroitin sulphate formed in the presence of 0.1 mM-4-methylumbelliferyl beta-xyloside contained 30-40% less sulphate than did chondrotin sulphate synthesized in the absence of xylosides. Similar experiments, with [3H]serine instead of [3H]glucose, suggested also a 20-30% decrease in chain length of the chondroitin sulphate; this was confirmed by direct gel filtration of labelled glycosaminoglycans on a calibrated column. Incorporation of [3H]glucose or [3H]serine was stimulated by serum and tri-iodothyronine in parallel with incorporation of sulphate. The changes seen in the total chondroitin sulphate were mirrored in the major proteoglycan fraction, purified by isopycnic centrifugation of salt-extracted proteoglycans. The labelling pattern of chondroitin sulphate from this proteoglycan indicated that decreased sulphation of chondroitin sulphate was largely due to the inferior ability of short polysaccharide chains to accept sulphate, with some direct interference with transfer of sulphate to all chains. The results also suggested that the action of serum factors on synthesis of proteochondroitin sulphate is exercised at the level of either protein synthesis or transport to the sites of initiation of polysaccharide synthesis.     

Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum-arrested and serum-stimulated cells. Addition of platelet-derived growth factor to serum-arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000-dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5-6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.