Proteoglycan synthesis in suspension cultures of Swarm rat chondrosarcoma chondrocytes and inhibition by exogenous hyaluronate

Conditions were established for short-term primary suspension culture of chondrocytes from the Swarm rat chondrosarcoma. Proteoglycan and hyaluronate synthesis on Day 0 to Day 2 in culture was investigated and compared with that for plated cultures. Incorporation of [35S]sulfate into proteoglycans was the same for both suspension and plated cultures. 35S-Proteoglycan synthesis decreased by about 80% between Days 0 and 1 irrespective of culture conditions. Suspension culture chondrocytes synthesized proteoglycans which were very similar to those made in plated cultures, with respect to hydrodynamic size, glycosaminoglycan, chain length, and composition. [3H]Hyaluronate synthesis accounted for 18 and 23% of the total 3H-glycosaminoglycans synthesized from [3H]glucosamine by suspension and plated cultures, respectively. Suspension culture chondrocytes responded to exogenous hyaluronate (1 mg/ml) by reducing their 35S-proteoglycan synthesis by about 50%. [3H]Hyaluronate synthesis was inhibited by 13% under these conditions. The inhibition was dependent on the concentration of exogenous hyaluronate and reached a plateau level within 2 h. Plated chondrocyte cultures showed little or no response to hyaluronate. Suspension cultures of chondrocytes were prelabeled with [3H]lysine and lysed, and a heavy membrane fraction (12,000g) was extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. A Sepharose-hyaluronate affinity gel was used to show that the extract contained hyaluronate binding 3H-labeled proteins and evidence was obtained suggesting that these came from the external face of the plasma membrane.     

Tunicamycin partially delays release of newly synthesized hyaluronate from Swarm rat chondrosarcoma chondrocytes

Tunicamycin (5-100 micrograms/ml) inhibits total [3H]hyaluronate synthesis in cultures of Swarm rat chondrosarcoma chondrocytes by approx. 15%. In agreement with previous results (Lohmander, L.S., Fellini, S.K., Kimura, J.H., Stevens, R.L. and Hascall, V.C. (1983) J. Biol. Chem. 258, 12280-12286) the relative decrease in [3H]hyaluronate radioactivity in the culture medium was greater than in the cell layer. Treated cultures show a concentration-related decrease in the proportion of medium 35S-labelled proteoglycans forming 'natural aggregates'. Pulse-chase experiments in cultures pretreated with tunicamycin (100 micrograms/ml, 13 h) showed that 30-40% of the total [3H]hyaluronate synthesized is released more slowly from these chondrocytes than from control culture chondrocytes. Release of some hyaluronate molecules may be delayed by 6 h or more. After a 24 h chase period almost all the [3H]hyaluronate is released from the cells. The proportion of 35S-labelled proteoglycans present as aggregates in the 24 h chase medium (57%) remained depressed compared to controls (81%), although the monomers could form aggregates if exogenous hyaluronate was added. Hyaluronate synthesized in the presence of tunicamycin has the same hydrodynamic size as control culture hyaluronate, as assessed by its sedimentation profile in CsSO4 gradients and its chromatographic profile on a dissociative Sephacryl S-1000 column.     

Effect of testicular hyaluronidase on hyaluronate synthesis by human skin fibroblasts in culture

The effect of hyaluronidase treatment on the incorporation of [3H]glucosamine into hyaluronate in human skin fibroblast cultures was investigated. Fourth passage cells in confluent cultures were treated with hyaluronidase from bovine tests, Streptomyces and leech in Dulbecco's minimum essential medium in the presence of 3% fetal calf serum. The medium was removed from the control (non-treated) and the treated cultures and the washed cell layers were incubated with [3H]glucosamine and [35S]sulfate. [3H]Hyaluronate was separated by DEAE Trisacyl chromatography and identified by specific enzymic assays. Hyaluronidase treatment induced an increase in the amount of labelled hyaluronate secreted into the medium and into the pericellular compartment. This amount reached a plateau with increasing enzyme concentration and with the time of treatment. Oligosaccharides derived from hyaluronate did not produce this effect. The maximal increase was about 3-fold, and was not inhibited by exogenous hyaluronate (25-100 micrograms/ml) or by oligosaccharides from hyaluronate. Cycloheximide (0.03 mM) inhibited hyaluronate synthesis by 18% or less in the control cells and by 50% in the hyaluronidase-pretreated fibroblasts. No significant difference was found in the hyaluronate synthase activity between control and treated cells, at 60 min following treatment, indicating the reversibility of the effect. The persistence of the stimulation required the presence of hyaluronidase. The treatment of cells with specific hyaluronidases (from Streptomyces and leech) or with testicular hyaluronidase did not modify the labelling of the sulfated glycosaminoglycans. The incorporation kinetics of the [3H]glucosamine into labeled hyaluronate and the increased amount of non-labelled hyaluronate determined by radiometric assay indicated a specific stimulation of hyaluronate synthesis in the hyaluronidase-pretreated fibroblast cultures.     

Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.     

Formation of proteoglycan aggregates in rat chondrosarcoma chondrocyte cultures treated with tunicamycin

Proteoglycan monomer and link protein isolated from the Swarm rat chondrosarcoma both contain glycosylamine-linked oligosaccharides. In monomer, these N-linked oligosaccharides are concentrated in a region of the protein core which interacts specifically with both hyaluronate and link protein to form proteoglycan aggregates present in cartilage matrix. Chondrocyte cultures were treated with tunicamycin to inhibit synthesis of the N-linked oligosaccharides, and the ability of the deficient proteoglycan and link protein to form aggregates was studied. Cultures were pretreated with tunicamycin for 3 h and then labeled with either [3H]mannose, [3H]glucosamine, [3H]serine, or with [35S]sulfate for 6 h in the presence of tunicamycin. Formation of link protein-stabilized proteoglycan aggregates in the culture medium was inhibited by up to 40% when the cells were treated with 3 micrograms of tunicamycin/ml, a concentration which inhibited 3H incorporation with mannose as a precursor by about 90%, but by only 15% with glucosamine as a precursor. When exogenous proteoglycan aggregate was added to the culture medium, however, it was found that both endogenous monomer and link protein synthesized in the presence of tunicamycin were fully able to form link-stabilized aggregates. This suggests that glycosylamine-linked oligosaccharides on monomer and on link protein are not necessary for their specific interactions with hyaluronate and with each other. Further, although tunicamycin did not inhibit net synthesis of hyaluronate, transfer of hyaluronate from the cell layer to the culture medium was retarded. This phenomenon accounted for most if not all of the decrease in the amount of proteoglycan which formed aggregates in the medium of cultures treated with tunicamycin.     

Absence of covalently linked core protein from newly synthesized hyaluronate.

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth

Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5'-nucleotidase are suggested as modulators of hyaluronate synthesis.     

Glycoprotein synthesis in explants of developing rabbit mammary gland in culture.

1. Explants of mammary glands of pregnant rabbits cultured in the absence of insulin, prolactin and cortisol incorporated [2-3H]mannose into lipid-linked mono- and oligo-saccharide and protein. 2. Inclusion of the hormones in the culture medium stimulated the incorporation of [2-3H]mannose into lipid-linked monosaccharide 4-fold, into lipid-linked oligosaccharide 4-fold and into protein 13-fold after 24 h in culture. 3. Addition of tunicamycin to the incubation medium completely inhibited the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and protein after an initial lag period of about 2h. Incorporation of this radiolabel into lipid-linked monosaccharide was increased 4-fold under these conditions. 4. Incorporation of [4,5-3H]leucine into protein was unaffected by the presence of tunicamycin. 5. Analysis of mannose-labelled protein by polyacrylamide-gel electrophoresis indicated that a major radiolabelled protein of apparent mol.wt. 65,000-70,000 was synthesized and approx. 70% of this protein appeared in the soluble fraction. 6. Glycosylation of the protein but not synthesis of its peptide backbone was sensitive to tunicamycin. 7. Possible origins of this glycoprotein synthetized when the tissue is stimulated to differentiate in culture are discussed.     

Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo

We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.     

Effects of vanadate on tyrosine phosphorylation and the pattern of glycosaminoglycan synthesis in rabbit chondrocytes in culture

The present study examined the effects of high doses of vanadate on glycosaminoglycan (GAG) synthesis and tyrosine phosphorylation in rabbit chondrocytes in confluent cultures. Although 6 microM vanadate increased the incorporation of [3H]glucosamine into chondroitin sulfate proteoglycans twofold, 40-60 microM vanadate suppressed this incorporation fourfold. Although 6 microM vanadate had little effect on [3H]glucosamine incorporation into hyaluronate, 40-60 microM vanadate increased this incorporation threefold. Chemical analyses confirmed that the increase in [3H]glucosamine incorporation into hyaluronate and the decrease in the incorporation into chondroitin sulfate proteoglycan correlated with increased hyaluronate content and decreased chondroitin sulfate content in the cell layers of vanadate-transformed cells. Chondrocytes exposed to 40-60 microM vanadate became typically transformed spindlelike cells. Furthermore, vanadate, at 6 and 60 microM, increased the overall level of phosphotyrosine by 8- and 31-fold, respectively, and 60 microM vanadate enhanced phosphorylation of many phosphotyrosine-containing proteins. These observations suggest that vanadate induces transformation-associated changes in the pattern of GAG synthesis when it induces excess phosphorylation on tyrosine in chondrocyte proteins.     

Stimulation of glycosaminoglycan production in murine tumors

Three types of murine tumors, B-16 melanoma, A-10 carcinoma, and S-180 sarcoma, were shown to contain elevated glycosaminoglycan (GAG) concentrations in vivo as compared to normal muscle or subcutaneous tissue. Hyaluronate was especially concentrated in the A-10 carcinoma, which contained approximately six times more hyaluronate than subcutaneous tissue and 18 times more than muscle. In all three tumors, chondroitin sulfates, especially chondroitin-4-sulfate, were present in higher concentrations than in the normal tissues. In culture, however, all three tumor cell lines produced less than 5% as much GAG as mouse fibroblasts, when measured by incorporation of [3H] acetate or by chemical analysis. Varying the culture passage number or the medium composition, ie, glucose, serum, and insulin concentrations, had little effect on GAG synthesis by the tumor cells. The low GAG levels in the tumor cell cultures were not due to hyaluronidase activity in their media. In an attempt to mimic possible host-tumor cell interactions that could account for the elevated GAG levels in vivo, tumor cells were cocultured with fibroblasts, but no stimulation above the amount made by the tumor cells alone plus that by the fibroblasts alone was observed. Conditioned media from the tumor cells, either dialyzed or not against fresh complete medium, had no effect on fibroblast GAG synthesis. Tumor extracts, however, were found to stimulate synthesis of hyaluronate by fibroblasts. Stimulation by extracts of A-10 carcinoma was greater than and additive to that of serum. The above results strongly suggest that GAG production in these tumors is in part regulated by host-tumor interactions.     

In vitro synthesis and secretion of glycoprotein by human mammary tissue

Summary Organ cultures of human surgical specimens can be used to investigate glycoprotein production in vitro under conditions in which three-dimensional tissue structures and cell-cell interactions resemble those present in vivo. In this report, an organ-culture system is used to investigate the synthesis, transport and release of glycoprotein by normal and benign hyperplastic human mammary epithelium. Autoradiography of explants pulse-labeled with individual glycoprotein precursors ([³H]glucosamine, [³H]fucose, [³H]acetylmanosamine) and maintained in organ culture for intervals up to 72hr revealed that glycoprotein is synthesized and then secreted by mammary epithelium. Incorporation of each isotope took place in the Golgi apparatus. Most of the newly synthesized glycoprotein, labeled with each of the three precursors, then was transported to apical cell surfaces and secreted into gland lumina. Observations were indistinguishable in normal and benign hyperplastic glands. Thus nonlactating human mammary epithelium exhibits a glycoprotein secretory activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [³H]glucosamine-labeled macromolecules released into the medium showed a group of glycoproteins with a molecular weight of 48,000±6,000 daltons plus high-molecular-weight glycosylated components at the top of gels. The nature of gp48 is not known, but similar molecular-weight glycoproteins also are released by surgical specimens of human mammary cancer maintained in organ culture. Z. A. T. received support from NCI Grant No. CA-14089.     

Increased hyaluronate synthesis is required for fibroblast detachment and mitosis.

Human-embryo fibroblasts were synchronized by means of colchicine and cytochalasin, and the production of hyaluronate was determined by [3H]glucosamine incorporation and ion-exchange chromatography. Cells arrested by colchicine synthesized small amounts of hyaluronate, whereas cells blocked by cytochalasin were stimulated in hyaluronate production. When the colchicine block was released, there was an increased synthesis of hyaluronate, which appeared first in the cellular fraction and was then shed into the culture medium. After release of the cytochalasin block, the hyaluronate production declined to that found with unsynchronized cells. A comparable increase of hyaluronate synthase activity was observed during mitosis. When hyaluronate synthesis was blocked by periodate-oxidized UDP-glucuronic acid, the cells were arrested in mitosis before rounding of cells. These results suggest that hyaluronate synthesis is required for detachment and rounding of cells during mitosis.     

Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts.

Hyaluronate synthase activity is localized exclusively in plasma-membrane fractions of cultured human skin fibroblasts. The enzyme activity of plasma membranes prepared from exponential-growth-phase cells was about 6.5 times that of stationary-growth-phase cells. Hyaluronate synthase from exponential-growth-phase cells exhibited lower Km and higher Vmax. values for both UDP-N-acetylglucosamine and UDP-glucuronic acid and higher rate of elongation of hyaluronate chains compared with the enzyme from stationary-growth-phase cells. Hyaluronate synthase exhibited an extremely short half-life, 2.2 h and 3.8 h respectively when cells were treated with cycloheximide and actinomycin D. The cell-growth-phase-dependent variations in hyaluronate synthase activity appear to be due to its high turnover rate as well as due to some post-translational modification of the enzyme protein as cells progress from early exponential to stationary growth phase. The isolated plasma membranes contained a protein (Mr approx. 450,000) that was selectively autophosphorylated from [gamma-32P]ATP in vitro in the presence of hyaluronate precursors in the reaction mixture and that also exhibited some hyaluronate-synthesis-related properties. The 32P-labelled protein isolated from plasma membranes of exponentially growing cells expressed an efficient UDP-[14C]glucuronic acid- and UDP-N-acetyl[3H]glucosamine-binding activity and was able to synthesize oligosaccharides (Mr 5000) of [14C]glucuronic acid and N-acetyl[3H]glucosamine residues. The corresponding protein of stationary-growth-phase cells, which expressed much higher nucleotide-sugar-precursor-binding activity, appeared to have lost its oligosaccharide-synthesizing activity.     

Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage.

The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.     

Organ culture of adult rat colonic mucosa on fibrin foam

A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37 degrees C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum, L-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmostphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.