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Biomedical subjects

M Yanagishita

Publications and source records attributed to M Yanagishita.

At least 19 recordsLinked to original sources
PubMed1995

Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells.

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.

Animals↗
PubMed1995

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures.

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.

Aggrecans↗
PubMed1995

Biosynthesis of proteoglycans and hyaluronic acid by rat oral epithelial cells (keratinocytes) in vitro.

Biosynthesis of complex carbohydrates, including sulfated glycoproteins, hyaluronic acid (HA), and proteoglycans (PGs), synthesized by rat oral epithelial cells (keratinocytes) in culture were studied by metabolic labeling protocols using [35S]sulfate and [3H]glucosamine in combination with differential enzymatic digestion and analytical gel filtration. The epithelial cells synthesized a major sulfated glycoprotein species with an apparent molecular size approximately 50 kDa, which accounted for approximately 46% of the total 35S incorporation. HA was a relatively minor component of 3H-labeled macromolecules (approximately 4% of the total 3H incorporation), and almost all of it was secreted into the medium. PGs accounted for approximately half of the 35S incorporation, of which about 30% was secreted into the medium and the remainder associated with the cell layer. The majority of PGs (75% of the secreted and 97% of the cell-associated) contained heparan sulfate (HS) and had an apparent molecular weight of approximately 150,000. Cell-associated HSPGs had a core protein of approximately 70 kDa with HS chains of approximately 64 kDa, while HSPG in the medium had a core protein of approximately 50 kDa with HS chains of the same average size as those of the cell-associated HSPG. Of the total cell-associated HSPGs, glycosylphosphatidyl inositol-anchored forms, plasma membrane intercalated forms and those associated with basolateral pericellular matrix accounted for approximately 3%, 56% and approximately 4% of the total, respectively. Approximately one third of the cell-associated HSPGs were intracellular components most likely generated through intracellular degradation processes following endocytosis. Cell surface HSPGs synthesized by keratinocytes may be involved in some biological roles such as the regulation of normal epithelial turnover and defense mechanisms involving interactions with various oral pathogens.

Animals↗
PubMed1995

Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.

Amino Acid Sequence↗
PubMed1995

Proteoglycans in haemodialysis-related amyloidosis.

Changes in extracellular matrices of articular tissue, intervertebral discs and systemic organs in patients with haemodialysis-related amyloidosis were investigated by immunohistochemical and biochemical examination of proteoglycans. Increased staining for chondroitin sulfate (CS) was detected in the amyloid deposits of all patients, ranging from early to advanced stages. Degenerative tissue changes around early-stage amyloid deposits in the intervertebral discs also showed positive staining for CS. Heparan sulfate (HS) was detected in amyloid deposits, especially in the synovial membrane. Biochemical analysis of connective tissues containing amyloid supported the immunohistochemical studies; CS was the major glycosaminoglycan species in these tissues, accounting for 55-81% of the total glycosaminoglycans. Although previous studies have stressed the importance of HS in amyloidogenesis, the present study showed that CS, which increased significantly in articular tissues associated with mechanical stress, also has a close relationship with amyloidogenesis.

Adult↗
PubMed1995

Mediation of human immunodeficiency virus type 1 binding by interaction of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41.

The mechanism of heparan sulfate (HS)-mediated human immunodeficiency virus type 1 (HIV-1) binding to and infection of T cells was investigated with a clone (H9h) of the T-cell line H9 selected on the basis of its high level of cell surface CD4 expression. Semiquantitative PCR analysis revealed that enzymatic removal of cell surface HS by heparitinase resulted in a reduction of the amount of HIV-1 DNA present in H9h cells 4 h after exposure to virus. Assays of the binding of recombinant envelope proteins to H9h cells demonstrated a structural requirement for an oligomeric form of gp120/gp41 for HS-dependent binding to the cell surface. The ability of the HIV-1 envelope to bind simultaneously to HS and CD4 was shown by immunoprecipitation of HS with either antienvelope or anti-CD4 antibodies from 35SO4(2-)-labeled H9h cells that had been incubated with soluble gp140. Soluble HS blocked the binding of monoclonal antibodies that recognize the V3 and C4 domains of the envelope protein to the surface of H9 cells chronically infected with HIV-1IIIB. The V3 domain was shown to be the major site of envelope-HS interaction by examining the effects of both antienvelope monoclonal antibodies and heparitinase on the binding of soluble gp140 to H9h cells.

Antibodies, Monoclonal↗
PubMed1993

Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures.

The viscoelastic properties of culture medium obtained from confluent 3T3-L1 preadipocytes, after differentiation with isobutyl-methylxanthine and dexamethasone, were studied with a rotational Couette viscometer. In close association with adipocyte differentiation, the culture medium showed gel-like properties, in concert with an increase in viscosity. This behavior vanishes after digestion by Streptomyces hyaluronidase or chondroitinase ABC, but not after application of collagenase, pronase, trypsin, DNase, or neuraminidase, or by treatment with EDTA or mercaptoethanol, indicating that the primary substance responsible for this behavior is hyaluronic acid. The material revealed a non-Newtonian behavior with an irreversible disruption of the network by shear force at high speeds. The viscosity of the medium, containing about 1 microgram/ml of hyaluronic acid, was calculated to be similar to that of a solution containing 1.7 mg high molecular weight hyaluronic acid per milliliter of stock culture medium. The comparison of rheological properties between the culture medium and solutions of hyaluronic acid indicated the possibility of a highly organized network in the culture medium that is more complicated than a simple interaction between homologous hyaluronic acid molecules. The non-Newtonian behavior depends on the hyaluronic acid concentration in the medium as well as on the length of exposure of the 3T3-L1 cells to the isobutyl-methylxanthine/dexamethasone mixture. The results point toward the possibility of interaction between hyaluronic acid and binding proteins.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Effects of MgCl2 on the release and recycling of heparan sulfate proteoglycans in a rat parathyroid cell line.

Divalent cations, such as Mg2+, Ba2+, and Co2+, are known to mimic the effects of Ca2+ in parathyroid cells, but it is not clear whether the mechanism of their action is the same as that of Ca2+. We have shown that extracellular Ca2+ concentration ([Ca2+]e) regulates the distribution and recycling of cell-surface heparan sulfate (HS) proteoglycans in a rat parathyroid cell line; at normal to high [Ca2+]e (e.g., 2 mM) HS proteoglycans are primarily localized intracellularly, while at low [Ca2+]e (0.05 mM) they are translocated to the cell surface and rapidly recycle (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C., 1990, J. Biol. Chem. 265, 13661-13668). We now show that a high concentration of Mg2+ (8 mM) reduces the amount of recycling HS proteoglycans in low [Ca2+]e. However, the primary effects of high Ca2+ and high Mg2+ on the recycling HS proteoglycans are different. High [Ca2+]e causes translocation of HS proteoglycans to intracellular compartments, while high Mg2+ stimulates cleavage of their core proteins and subsequent shedding of HS proteoglycans into the medium, thereby depleting the recycling molecules. However, high Mg2+ does not induce shedding of HS proteoglycans in high [Ca2+]e. The effects of Ba2+ and Co2+ were similar to those of Mg2+, but Sr2+ showed no significant effects on HS proteoglycan translocation. Otherwise, 8 mM Mg2+ did not alter biosynthesis or intracellular catabolism of HS proteoglycans. These observations suggest that the recycling of HS proteoglycans in parathyroid cells is sensitive only to [Ca2+]e, whereas several other divalent cations can deplete the recycling HS proteoglycans by a distinctly different mechanism. Thus, the mechanism by which Ca2+ regulates the amounts of the recycling HS proteoglycans may be more physiological and play a functional role in parathyroid cells.

Animals↗
PubMed1992

Metabolic pathways of heparan sulfate proteoglycans in a rat parathyroid cell line.

The distribution of heparan sulfate (HS) proteoglycans in clonal rat parathyroid cells is regulated by the extracellular Ca2+ concentration, which is a principal factor for parathyroid cell function (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C. (1990) J. Biol. Chem. 265, 13661-13668). Increasing the concentration of extracellular Ca2+ in the physiological range redistributes HS proteoglycans from the cell surface to an intracellular compartment. We have now examined effects of the extracellular Ca2+ concentration on the metabolism of the HS proteoglycans in detail using [35S]sulfate metabolic labeling-chase experiments. Two distinct metabolic pathways were demonstrated: (i) the intracellular generation of HS chains from HS proteoglycans in prelysosomal compartments followed by their release into the medium (pathway 1), and (ii) intracellular generation of HS oligosaccharides from HS chains in prelysosomal compartments, which are eventually degraded into free sulfate in lysosomes (pathway 2). The HS oligosaccharides were exclusively present within the cells, whereas HS chains were found primarily in the medium. The cells do not internalize either HS proteoglycans or HS chains from the medium. These observations indicate that these two degradation pathways are independent. In addition to these pathways, approximately 15% of the HS proteoglycans were released into the medium as a proteoglycan form. Treatment of cells with chloroquine, a lysosomotropic agent, did not affect generation of HS chains but inhibited conversion of HS chains to HS oligosaccharides or to free sulfate and resulted in the release of HS chains from the cells. The drug did not affect metabolic pathway 1. The extracellular Ca2+ concentration did not alter these intracellular degradation pathways for HS proteoglycans in the parathyroid cells. Thus, extracellular Ca2+ appears to regulate only the distribution of HS proteoglycans between the cell surface and intracellular compartments, and the process of cycling between these compartments when extracellular Ca2+ is low.

Animals↗
PubMed1992

Recycling of transferrin receptors and heparan sulfate proteoglycans in a rat parathyroid cell line.

We examined recycling of heparan sulfate (HS) proteoglycans and transferrin receptor (Tf-R) in a rat parathyroid cell line. While extracellular Ca2+ concentration ([Ca2+]e) regulates the recycling of HS proteoglycans in parathyroid cells, such that HS proteoglycans only recycle when [Ca2+]e is lowered below physiological levels, recycling of Tf-R occurs equally well both in 0.05 mM (low) and 2 mM (high) [Ca2+]e. Inhibiting endocytosis chemically with phenylarsine oxide or at low temperature (4 degrees C) did not abolish the effects of changing [Ca2+]e on HS proteoglycans in the recycling compartment even though transport of HS proteoglycans from the Golgi complex to the cell surface was inhibited in low [Ca2+]e. Microtubules are not involved in the recycling of HS proteoglycans or of Tf-R since nocodazole did not affect these processes. Inhibiting the increase of intracellular Ca2+ by an intracellular Ca2+ chelator sustained recycling of HS proteoglycans even in the presence of high [Ca2+]e. These observations show that the exocytosis pathway of HS proteoglycans in the recycling compartment is specifically regulated by [Ca2+]e, whereas that for constitutive secretion is not. Therefore, the recycling of HS proteoglycans may be directly related to some functions of parathyroid cells regulated by [Ca2+]e. Although the mechanism by which [Ca2+]e regulates the exocytosis and recycling of HS proteoglycans is uncertain, it is suggested that an increase of intracellular Ca2+ is necessary, but not necessarily sufficient, for inhibiting their exocytosis.

Affinity Labels↗
PubMed1992

Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP).

The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.

Affinity Labels↗
PubMed1992

Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.

Animals↗
PubMed1992

Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways.

Rat ovarian granulosa cells synthesize two distinct species of plasma membrane-intercalated heparan sulfate (HS) proteoglycans; glycosylphosphatidylinositol (GPI)-anchored and core protein-intercalated HS proteoglycans. Both species of HS proteoglycans are primarily localized on the plasma membrane. Cell surface localization of GPI-anchored and protein-intercalated HS proteoglycans can be determined by their accessibility to exogenously added phosphatidylinositol-specific phospholipase C (PI-PLC) and trypsin, respectively. Kinetic parameters for the processes involving their transfer from the Golgi to the cell surface, endocytosis and secretion, and the modes of intracellular degradation were determined by metabolic labeling experiments using [35S]sulfate and various chase protocols in combination with the use of PI-PLC and trypsin in rat ovarian granulosa cells. The experiments demonstrated that (i) both HS proteoglycan species are transferred from the Golgi to the cell surface with an average transit time of approximately 12 min. (ii) GPI-anchored HS proteoglycans are endocytosed with a t1/2 approximately 3 h, without being shed into the medium, and they are rapidly degraded, t1/2 approximately 25 min, without generating recognizable degradation intermediates. (iii) Protein-intercalated HS proteoglycans are partly (approximately 30%) shed from the cell surface into the medium and the remaining approximately 70% are endocytosed with a t1/2 approximately 4 h. After endocytosis, they undergo a slow (t1/2 approximately 4 h) stepwise degradation generating distinct HS oligosaccharides as degradation intermediates. These results indicate that the GPI-anchored and the protein-intercalated HS proteoglycans have distinct secretory, endocytotic, and intracellular degradation pathways probably due to the differences in their anchor structures.

Animals↗
PubMed1992

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.

Aging↗
PubMed1992

Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle.

Mural and cumulus granulosa cells synthesize hyaluronic acid (HA) and expand in vitro in response to follicle-stimulating hormone and a soluble factor(s) produced by fully grown oocytes. In the present study we examined HA synthesis and extracellular matrix organization by the two cell populations in vivo during the preovulatory period. After injection of human chorionic gonadotropin into pregnant mares' serum gonadotropin-primed animals, a progressive increase in HA synthesis was observed by the cumulus cell-oocyte complex (COC), and by the mural granulosa cells adjacent to the antrum (antral granulosa cells). The outermost layers of mural granulosa cells (peripheral granulosa cells) did not synthesize HA. Net HA synthesis was approximately 4 pg/cell for COCs isolated after full expansion induced either in vivo or in vitro, whereas the total HA content and cell number in the ovulated COC (approximately 11 ng HA and approximately 3000 cells per COC) were about threefold higher than for COCs expanded in vitro (approximately 4 ng HA and approximately 1000 cells per COC). The increased cell content of ovulated COCs appears to be primarily the result of inclusion of proximal mural granulosa cells which synthesize HA in response to the oocyte factor(s) and become incorporated in the expanded COC extracellular matrix mass. Media conditioned by oocytes enclosed in the cumulus cell mass (intact COCs) contained only 10-20% of the HA-stimulatory activity of media conditioned by an equal number of isolated oocytes when tested on mural granulosa cell cultures. Further, HA-stimulatory activity of media conditioned by isolated oocytes was dramatically reduced (approximately 70%) by preincubation for 5 hr with cumulus cells compared to preincubation in the absence of cells. The results suggest that differences in HA synthesis between subregions of membrana granulosa depend on a diffusion gradient of the oocyte factor(s).

Animals↗
PubMed1991

Proteoglycans synthesized by an osteoblast-like cell line (UMR 106-01).

The proteoglycans synthesized by an osteoblast-like cell line of rat origin (UMR 106-01) were defined after biosynthetic labelling with [35S]sulphate and [3H]glucosamine. Newly synthesized labelled proteoglycans were characterized by differential enzymic digestion in combination with analytical gel filtration and SDS/PAGE. UMR 106-01 cells were found to synthesize three major species of proteoglycan: a large chondroitin sulphate proteoglycan of Mr approximately 1 x 10(6), with a core protein of Mr approximately 350,000-400,000; a small chondroitin sulphate-containing species of Mr approximately 120,000 with a core protein of Mr 43,000; and a heparan sulphate proteoglycan of Mr approximately 150,000, with a core protein of Mr approximately 80,000. Over 70% of the newly synthesized intact proteoglycan species are associated with the cell layer of near-confluent cells; however, accessibility to trypsin digestion suggests an extracellular location. Chemical characteristics of the proteoglycans and preliminary mRNA hybridization indicate that the small chondroitin sulphate proteoglycan is probably PG II (decorin). The large chondroitin sulphate proteoglycan is most likely related to a hyaluronate-aggregating species from fibroblasts (versican), and the heparan sulphate proteoglycan bears striking similarities to cell-membrane-intercalated species described for a number of cell types.

Animals↗