The effect of chronic ethanol consumption on temperature-dependent physical properties of liver mitochondrial membranes

We have reported that the monovalent ionophore monensin causes undersulfated chondroitin sulfate biosynthesis in cultured chondrocytes. In order to clarify the mechanism of this diminished sulfation, we have measured the rate of incorporation of sulfate into chondrocytes and assayed the cellular ATP levels. We have also measured sulfatase activity, the incorporation of ³⁵SO₄ into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and endogenous sulfotransferase activity in the cell-free extracts. We find that: (1) The incorporation of ³⁵SO₄ into the free sulfate pool in chondrocytes was not inhibited by monensin. (2) The ATP levels of monensin-treated chondrocytes were the same as control cells. (3) There was no sulfatase activity in both control and monensin-treated chondrocytes. (4) Enzymatic analyses revealed that ³⁵SO₄ incorporation into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and subsequent sulfotransferase activity were not inhibited in the presence of monensin. At present the most tenable hypothesis to account for monensin causing undersulfated chondroitin sulfate synthesis is that the ionophore impairs the access of proteoglycans to the sulfotransferases in the luminal walls of the Golgi structures.     

Fine structure and molecular content of human chondrocytes encapsulated in alginate beads

Preservation of the chondrocytic phenotype in vitro requires a 3D (three‐dimensional) culture model. Diverse biomaterials have been tested as scaffolds for culture of animal chondrocytes; however, to date, none is considered a gold standard in regenerative medicine. Here, we studied the fine structure and the GAGs (glycosaminoglycans) content of human chondrocytes encapsulated in alginate beads by using electron microscopy and radioactive sulfate [³⁵S] incorporation, respectively. Cells were obtained from human cartilage, encapsulated in alginate beads and cultured for 28 days. [³⁵S]Na₂SO₄ was added to the culture media and later isolated for quantification of the sulfated GAGs found in three compartments: IC (intracellular), IB (intra‐bead) and EB (extra‐bead). Round cells were seen isolated or forming small groups throughout the alginate. Human chondrocytes presented the features of active cells such as euchromatic nuclei, abundant RER (rough endoplasmic reticulum) and many transport vesicles. We observed an extracellular matrix rich in collagen fibres and electrondense material adjacent to the cells. Most of the GAGs produced (74%) were found in the culture medium (EB), indicating that alginate has a limited capacity to retain the GAGs. CS (chondroitin sulfate), the major component of aggrecan, was the most prominent GAG produced by the encapsulated cells. Human chondrocytes cultured in alginate can sustain their phenotype, confirming the potential application of this biomaterial for cartilage engineering.     

Growth modulation and proteoglycan turnover in cultured mesangial cells

Proliferation of mesangial cells is a common feature of renal disease, and conditioned media from glomerular epithelial and endothelial cells have been found to contain heparin-like molecules that suppress proliferation of rat mesangial cells (RMC). We have partially characterized the glycosaminoglycans that are labeled with ³⁵SO₄^2? by RMC in culture at early passage and examined their ability to inhibit mitogenic stimulation of the cells. Four chondroitin/dermatan sulfate proteoglycans (CS/DSPG) were identified, the largest and smallest of which (K_d of 0.04 and 0.26 on Superose 6) were retained in the cell layer while the other two (K_d = 0.17 and 0.22) were secreted into the medium. Heparan sulfate proteoglycans (HSPG) with K_d values of 0.09, 0.13, and 0.39 were minor components of the cell layer, while a single heparan sulfate (K_d = 0.17) was recovered from the medium. After 16 h of labeling in serum-free medium, about 60% of macromolecular ³⁵S was cell-associated and 40% was in the medium. Cell-associated label consisted of 7% CS/DSPG, 9% HSPG, and 84% free glycosaminoglycan chains (mostly CS/DS), whereas the medium contained 52% CS/DSPG, 17% HSPG, and approximately equal amounts of free HS and CS/DS chains. Bovine lung heparin (1 μg/ml) decreased by 45% the incorporation of [³H]-thymidine into DNA after release of serum-starved RMC from growth arrest. Heparin acted prior to the G₁/S interface; arrest of the cells in early S phase with aphidicolin abrogated the heparin response. The endogenous HSPGs had a slight antimitogenic effect on the RMC, but heparan sulfate chains from both the medium and cell layer had a potent effect. On an equivalent mass basis, only the free glycosaminoglycan chains were more potent than heparin in this regard, decreasing thymidine incorporation by over 90% when present at 1 μg/ml. These results demonstrate that heparan sulfate glycosaminoglycans derived from mesangial proteoglycans are potential negative autocrine growth regulators. Proteoglycan metabolism releases these soluble heparan sulfate chains, determining the level of this activity. © 1994 wiley-Liss, Inc.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding

Liver endothelium has been reported to possess membrane receptors for the iron-binding protein transferrin (Tf). Similarly, the core protein of proteoglycans (PG) associated with cell membrane in many cell systems can bind Tf. To find out if membrane-associated proteoglycans can explain Tf-binding ability of liver endothelium, we investigated the synthesis and distribution of proteoglycans by isolated, cultured liver capillary endothelium. Cells were isolated and cultured for 48 h in sulfate-free medium and pulse-labeled with 35SO4. The relative distribution of 35SO4-labeled macromolecules, determined in the extracellular (EC), membrane-associated (MA), and intracellular (IC) pools, was respectively 74, 15, and 10%. Membrane-associated proteoglycan (MA-PG) was further purified by ion exchange and gel chromatography. Glycosaminoglycan (GAG) chain characterization indicated about 78% chondroitin sulfate, 7% dermatan sulfate, and about 14% heparan sulfate (HS). Similar GAG chain characterization was made for PG in the EC and IC pools. Transferrin-binding ability of MA-PG was studied by affinity column chromatography, using CNBr-activated sepharose bound to transferrin. About 15% of the labeled MA-PG was specifically bound to Tf-affinity column and could be eluted by excess soluble Tf. This proportion was similar to the proportion of HS in the total membrane-associated pool. Moreover, the eluted labeled material was susceptible to pretreatment with heparitinase, confirming its HS nature. We conclude that the transport capillary endothelium of the liver can synthesize HS proteoglycans which are membrane-associated and this MA-HS pool can bind transferrin. The finding may provide a molecular basis for transferrin binding to liver endothelium and may explain the subsequent transendothelial transport of iron-transferrin complexes into the liver.     

Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium

The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when ³H-glucosamine is used to label GAG as when ³⁵SO₄ is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium.     

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.     

Control of corneal differentiation by extracellular materials. Collagen as a promoter and stabilizer of epithelial stroma production

The primary stroma of the cornea of the chick embryo consists of orthogonally arranged collagen fibrils embedded in glycosaminoglycan (GAG) produced by the epithelium under the early inductive influence of the lens. The experiments reported here were designed to test whether or not the collagen of the lens basement lamina is capable of stimulating corneal epithelium to produce primary stroma. Enzymatically isolated 5-day-old corneal epithelia were grown for 24 hr in vitro in the presence of ³⁵SO₄ or proline-³H on various substrata. Epithelia cultured on lens capsule synthesized 2.5 times as much GAG (as measured by incorporation of label into CPC precipitable material) and almost 3 times as much collagen (assayed by hot TCA extraction or collagenase sensitivity) as when cultured on Millipore filter or other noncollagenous substrata. A similar stimulatory response was observed when epithelium was combined with chemically pure chondrosarcoma collagen, NaOH-extracted lens capsule, vitreous humor, frozen-killed corneal stroma or cartilage, or tendon collagen gels; in the latter case, the magnitude of the effect can be shown to be related to concentration of the collagen in the gel. All of the collagenous substrata stimulate not only extracellular matrix production, but also polymerization of corneal-type matrix, as judged by ultrastructural criteria and by the association of more radioactivity with the tissue than the medium. Since purified chondrosarcoma collagen is as effective as lens capsule, the stimulatory effect on collagen and GAG synthesis by corneal epithelium is not specific for basal lamina (lens capsule) collagen.     

Endocytosis and degradation of serglycin in liver sinusoidal endothelial cells

We have previously reported that liver sinusoidal endothelial cells (LSECs) are responsible for the clearance of monocyte chondroitin sulfate proteoglycan serglycin from the circulation (øynebråten et al.(2000) J. Leukocyte Biol. 67; 183–188). The aim of the present study was to investigate the kinetics of degradation of endocytosed serglycin in primary cultures of LSECs. The final degradation products of serglycin labelled biosynthetically in the glycosaminoglycan (GAG) chains with [³H] in the acetyl groups of N-acetyl galactosamine residues, [¹⁴C] in the pyranose rings, or [³⁵S] in the sulfate groups were identified as[³H]-acetate, [¹⁴C]-lactate and [³⁵S]-sulfate. Comparison of the rate of release of degradation products from the cells after endocytosis of serglycin labelled chemically with ¹²⁵I in the tyrosine residues, or biosynthetically with [³⁵S] or [³H] in the sulfate or acetyl groups, respectively, showed that ¹²⁵I appeared more rapidly in the medium than [³⁵S]-sulfate and [³H]-acetate. Judging from the speed of appearance of free ¹²⁵I both intracellularly and in the medium, the core protein is degraded considerably more rapidly than the GAG chains.Desulfation of the GAG chains starts after the GAG chains are released from the core protein. Generation of lactate and acetate as the final products from degradation of the carbon skeleton of the GAG chains indicates that catabolism of endocytosed macromolecules in LSECs proceeds anaerobically.     

Biosynthesis of glycosaminoglycans in the developing retina

The glycosaminoglycans of neural retinas from 5-, 7-, 10-, and 14-day chick embryos were labeled in culture with [³H]glucosamine and ³⁵SO₄, extracted, and isolated by gel filtration. The incorporation of label per retina into glycosaminoglycans increased with embryonic age, but that per cell and per unit weight of uronic acid decreased. Specific enzyme methods coupled with gel filtration and paper chromatography demonstrated that [³H]glucosamine incorporation into chondroitin sulfate increased between 5 and 14 days from 7 to 34% of the total incorporation into glycosaminoglycans. During this period, incorporation into chondroitin-4-sulfate increased relative to that into chondroitin-6-sulfate. Between 5 and 10 days, incorporation into heparan sulfate showed a relative decline from 89 to 61%. Incorporation into hyaluronic acid always represented less than 2% of the total. A twofold greater increase in galactosamine concentration than in glucosamine concentration in the glycosaminoglycan fraction between 7 and 14 days supports the conclusion that chondroitin sulfate was the most rapidly accumulating glycosaminoglycan. ECTEOLA-cellulose chromatography revealed a heterogeneity in the size and/or net charge of chondroitin sulfate and heparan sulfate. We conclude that incorporation of exogenous precursors into glycosaminoglycans in the chick retina decreases relative to cell number as differentiation progresses from a period of high mitotic activity to one of tissue specialization, and that it is accompanied by a net accumulation of glycosaminoglycan and a change in the pattern of its synthesis.     

Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42− uptake and expression of a band 3 like protein in the marine sponge,Microciona prolifera

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of ³⁵SO₄^2? uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in ³⁵SO₄^2? uptake and incorporation which could be greatly augmented in the presence of Ca²⁺/Mg²⁺. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa ³⁵SO₄^2? sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.     

Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-beta (TGF-beta)

The effect of transforming growth factor-beta (TGF-beta, 1 ng/ml) on proteoglycan synthesis by rabbit articular chondrocytes in culture was studied in the presence of fetal bovine serum. Exposure of confluent cells for 24 h to the factor resulted in a marked increase of 35S-labeled sulfate incorporation in the newly synthesized proteoglycans (PG), as estimated by glycosaminoglycan (GAG) radioactivity (+58%). The onset was observed 6 h after addition of the factor but was significant after 12 h. TGF-beta also enhanced the uptake of [35S]sulfate by chondrocytes, but had no effect on the release of PG by these cells. The effect of TGF-beta on the distribution of PG between the medium and the cell layer was shown to be dependent on the serum concentration in the medium: the relative proportion of cell-layer associated GAG of TGF-beta-treated cells decreased with increasing concentration of fetal bovine serum. The proportion of aggregated PG, the hydrodynamic size of PG monomers and GAG chains were not modified by TGF-beta, but the relative distribution of disaccharides 6- and 4-sulfate in GAG chains was altered by the factor: the proportion of chondroitin 6-sulfate (C6S) was decreased while that of chondroitin 4-sulfate (C4S) was augmented in presence of TGF-beta, leading to a decrease of the ratio C6S/C4S (-11 to -22%, P less than 0.01). The present study indicates that TGF-beta promotes the synthesis of a modified extracellular matrix in cultured articular chondrocytes. This mechanism could be relevant to some aspects of cartilage repair in osteoarticular diseases.     

Biosynthesis of chondroitin sulfate side chains and hyaluronate time-course studies with cartilage slices of calf ribs under different conditions

The time course of double labeling with ³⁵SO₄²⁻ and [³H]glucosamine was followed in a semi-in vitro system of cartilage slices from calf ribs whose chondroitin sulfate peptide pool consistsof (A) <1% of very short undersulfated side chains of <10 disaccharide units length, (B) 3–5% of short undersulfated longer side chains (16 to 25 disaccharide units), (C) 3–5% of short, slightly oversulfated side chains (16–23 dissacharide units, very probably containing some dermatan sulfate), (D) the bulk material (74–82% of total uronate) of longest, slightly undersulfated or equally sulfated side chains (22–42 disaccharide units).After 10 min incubation rapid chain elongation with [³H]glucosamine and prelabeling with ³⁵SO₄²⁻ of endogenous acceptors are apparent. Chains of type A exhibit highest specific radioactivities. During 30–60 min incubation it is mainly chains of type B that show highest specific radioactivities, after 90 min chains of type C. On the after hand, chains of type D always incorporated the highest total amount of both precursors. Preincubation of slices for 40 min at 37°C strongly enhances labeling rates of all types whilst preincubation for 40 min in an ice-bath enhances mainly ³⁵SO₄²⁻ labeling of types A and B.After 10 min preincubation followed by ³⁵SO₄²⁻ labeling for 60 min a decrease of radioactivity of type A and a distinct increase with type B are observed during the post incubation period. After pulse chase experiment type B exhibit highest specific radioactivities. The data make it evident that under-sulfated short chondroitin sulfate side chains from very rapidly in a well organised manner and grow, by elongation and proceeding sulfation processes, to longer higher sulfated chains.The labeling of the hyaluronate pool is about half of that of the chondroitin sulfate pool after a lag phase of 10 min. The latter increases linearly after 35–45 min incubation time. However, after preincubation and chase experiments the hyaluronate pool is more highly labeled. The data indicate different precursor pools of both biosynthesis mechanisms, probably located in different cell compartments and/or different cartilage cells.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the α and β subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [³⁵S]sulfate was also incorporated into several other proteins in addition to LH. The location of ³⁵SO₄^2? in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.     

The effect of prostaglandins on cultured lapine articular chondrocytes

The effect of 8 prostaglandins (PG) on growth and sulfate incorporation by monolayer and spinner-cultured rabbit articular chondrocytes has been measured. PGA₁, PGB₁, PGE₁ and PGE₂ reduced synthesis of sulfated glycosaminoglycans (GAG) but the PGF series did not. PGA₁ was the most potent, being effective at a concentration of 2.5 μg/ml [6.8 μM] while the others required 25 μg/ml. These compounds had no effect on degradation of GAG. All 8 PGs augmented growth slightly but significantly at 2.5 μg/ml. At the higher concentration, PGA₁ was highly cytotoxic, and PGB₁ as well as PGE₂ reduced cell growth. The cytotoxicity of PGA₁ was also observed in two additional types of cultured connective tissue cells, but the inhibition of sulfated-GAG synthesis by PGA₁ and PGB₁ was confined to the chondrocytes. The response of cultured chondrocytes to exogenous PGs, albeit at apparently unphysiologically high concentrations, together with other evidence, suggests that these compounds may conceivably play a direct role in cartilage metabolism in vivo.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Effects of colchicine on sulfated glycosaminoglycan production and cell detachment in pre-capillary pulmonary endothelial cells

The effects of colchicine on the morphology, substrate adhesiveness, and production of glycosaminoglycan (GAG) macromolecules by cultured pre-capillary pulmonary endothelial cell were studied. Colchicine-treated cells demonstrated altered morphology and decreased substrate adhesiveness compared to untreated cells. In addition, [35S]sulfate incorporation into glycosaminoglycans was decreased 33% after treatment with colchicine. Spectrophotometric measurement of total cellular GAG revealed a similar GAG reduction in colchicine-treated cells. The composition of [35S]sulfate radiolabelled GAG was similar in cultures with and without colchicine, consisting of approximately 56% chondroitin sulfate and the remainder heparin/heparan sulfate. The results indicate that colchicine influences the biological behavior of pre-capillary endothelial cells, in part by altering the amount of glycosaminoglycan molecules produced.     

Effect of beta-xylosides on synthesis of cartilage-specific proteoglycan in chondrocyte cultures.

Monolayer cultures of embryonic chick chondrocytes were incubated with ³⁵SO₄ in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free chondroitin sulfate chains were measured following gel filtration on Sephadex G-200. Synthesis of β-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. The amount of core protein was determined from equivalent numbers of β-xyloside-treated and untreated cells by a radioimmune assay. Similar amounts of core protein were found in both types of cultures, indicating that decreased synthesis of cartilage-specific core protein is not responsible for the observed decrease in overall chondroitin sulfate proteoglycan production.