Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.

Abstract. Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonaily defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5 % in HDM alone.

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin . Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy . Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix . Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated . Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation .Recently, we described a protein that was purified from fetal epiphyseal cartilage (1), that exists as a 70,000-mol-wt dimer of 35,000-mol-wt subunits. The protein binds strongly to hydroxyapatite and increases in concentration at the time when the secondary center of ossification appears in the epiphysis. This protein is unrelated to any other known cartilage or bone proteins (1). We prepared a monospecific antiserum to this protein and have now demonstrated immunohistochemically that it is intimately associated with the major calcification ofcartilage matrix that occurs in the lower hypertrophic zone of the growth plate . These observations are described in this article. They present the basis for a new understanding of cartilage calcification . We have called the protein chondrocalcin in view of its calcium-binding properties (P. Hauschka, H. V. Choi, and L. C. Rosenberg, unpublished results), its affinity for hydroxyapatite (1), and its presence in the calcified cartilage of growth plate, which is shown here. MATERIALS AND METHODSIsolation of Chondrocalcin and Other Molecules : Bovine fetal epiphyseal cartilage from the third trimester was extracted with 4 M guanidine hydrochloride as described previously (1). The extract was dialyzed to associative conditions and centrifuged with cesium chloride to produce density gradient fractions A 1 (high density) to A6 (low density) . Chondrocalcin was isolated from fractions AS and A6 by diethylaminoethyl cellulose chromatography followed by affinity chromatography with affi-gel blue and hydroxyapatite . T...

We aim to present phylogenetic major groups within the subfamily Cyperoideae (Cyperaceae) on the basis of three molecular data sets; nuclear ribosomal internal transcribed spacer and 5.8S ribosomal RNA region, the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit gene, and trnL intron and trnL-F intergenic spacer. Three molecular data and two combined data sets were used to obtain robust and detailed phylogenetic trees by using maximum parsimony and Bayesian inference, respectively. We analyzed 81 genera and 426 species of Cyperaceae, including Korean species. We suggest one early diverged group (EDGs), and two major clades (FAEC and SDC) within the subfamily Cyperoideae. And the clade EDGs comprises six tribes (Schoeneae, Bisboeckelereae, Sclerieae, Cryptangieae, Trilepideae, and Rhynchosporeae) at the basal nodes of Cyperoideae. The FAEC clade (posterior probability [PP]/bootstrap value [BS] = 1.00/85) comprises four tribes (Fuireneae, Abildgaardieae, Eleocharideae, Cypereae), and the SDC clade (PP/BS = 1.00/86) comprises three tribes (Scirpeae, Dulichieae, Cariceae). These three clades used for phylogenetic groups in our study will be useful for establishing the major lineage of the sedge family. The phylogeny of Korean sedges was also investigated within the whole phylogeny of Cyperaceae. The 20 genera of Korean sedges were placed in 10 tribes forming 14 clades.

Abstract. Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KSPGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was adsorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind.Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 313 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF. When a series of complementary cell-binding fragments generated from pFN by thermolysin digestion and subsequent affinity chromatography (Castellani, P., A. Siri, C. Rosellini, E. Infusini, L. Borsi, and L. , J. Cell Biol., 103:1671-1677 were tested, a graded response to inhibition by DS-PGs was observed revealing the proximity of the cryptic GAGbinding domain to the cell-binding domain of the fibronectin molecule.All of these results taken...