Effective doses from panoramic radiography and CBCT (cone beam CT) using dose area product (DAP) in dentistry.

OBJECTIVES: We compared the effective dose from panoramic radiography with that from cone beam CT (CBCT) using dose area product under adult and child exposure conditions. METHODS: The effective doses of the cephalo, panorama, implant and dental modes of Alphard 3030 (Asahi Roentgen Ind., Co. Ltd, Kyoto, Japan) CBCT and the Jaw, Wide, Facial and temporomandibular joint modes of Rayscan Symphony (RAY Co., Ltd, Hwaseong, Republic of Korea) CBCT were compared with those of CRANEX(®) 3+ CEPH (Soredex Orion Corporation, Helsinki, Finland) panoramic radiography equipment under adult and child exposure conditions. Each effective dose was calculated using a conversion formula from dose area product meter measured values (VacuTec Messtechnik GmbH, Dresden, Germany). The conversion formulae used were suggested by Helmrot and Alm Carlsson and Batista et al, and they were applied with the tube voltage taken into consideration. RESULTS: The maximum effective doses from the Alphard 3030 and Rayscan Symphony were 67 and 21 times greater than that from panoramic radiography, respectively. The ratios of the effective dose under the child setting to that under the adult condition were 0.60-0.62 and 0.84-0.95, and the maximum differences in effective doses between the adult and child exposure settings were equivalent to 27 and 4 times greater than a panoramic examination in the Alphard 3030 and Rayscan Symphony, respectively. CONCLUSIONS: The effective CBCT doses were higher than those of panoramic radiography. The differences in effective doses between the adult and child CBCT settings were dependent on equipment type and exposure parameters. Therefore, adequate mode selection and control of exposure as well as further research are necessary to minimize the effective dose to patients, especially for radiosensitive children.

Characterization of epiphycan, a small proteoglycan with a leucine-rich repeat core protein.

The epiphysis of developing bones is a cartilaginous structure that is eventually replaced by bone during skeletal maturation. We have separated a dermatan sulfate proteoglycan, epiphycan, from decorin and biglycan by using dissociative extraction of bovine fetal epiphyseal cartilage, followed by sequential ion-exchange, gel permeation, hydrophobic, and Zn2+ chelate chromatographic steps. Epiphycan is a member of the small leucine-rich proteoglycan family, contains seven leucine-rich repeats (LRRs), is related to osteoglycin (osteoinductive factor) (Bentz, H., Nathan, R. M., Rosen, D. M., Armstrong, R. M., Thompson, A. Y., Segarini, P. R., Mathews, M. C., Dasch, J., Piez, K. A., and Seyedin, S. M. (1989) J. Biol. Chem. 264, 20805-20810), and appears to be the bovine equivalent of the chick proteoglycan PG-Lb (Shinomura, T., and Kimata, K. (1992) J. Biol. Chem. 267, 1265-1270). The intact proteoglycan had a median size of approximately 133 kDa. The core protein was 46 kDa by electrophoretic analysis, had a calculated size of 34,271 Da, and had two approximately equimolar N termini (APTLES ... and ETYDAT ... ) separated by 11 amino acids. There were at least three O-linked oligosaccharides in the N-terminal region of the protein, based on blank cycles in Edman degradation and corresponding serine or threonine residues in the translated cDNA sequence. The glycosaminoglycans ranged in size from 23 to 34 kDa were more heterogeneous than those in other dermatan sulfate small leucine-rich proteoglycans and were found in the acidic N-terminal region of the protein core, N-terminal to the LRRs. A four-cysteine cluster was present at the N terminus of the LRRs, and a disulfide-bonded cysteine pair was present at the C terminus of the protein core. The seventh LRR and an N-linked oligosaccharide were between the two C-terminal cysteines. An additional potential N-glycosylation site near the C terminus did not appear to be substituted at a significant level.

The self-association of biglycan from bovine articular cartilage.

Biglycan is a small dermatan sulfate proteoglycan present in the extracellular matrix of a variety of connective tissues. Sedimentation velocity and equilibrium studies were carried out to determine the monomer molecular weight of biglycan in denaturing solvents and to define the oligomeric states of biglycan in physiologic solvents in the presence and absence of Zn2+. In 6 M guanidine chloride, biglycan is a monomer with s0(20,w) = 2.9 S and Mz = 93,100 (where Mz is z-average molecular weight). In 0.15 M NaCl, 50 mM Tris, pH 7.5, in the absence of divalent metal ions, and at concentrations above 1 mg/ml, biglycan is predominantly dimer (s0(20,w) = 4.8 S). Under these same conditions in solvent containing 5 mM Zn2+, biglycan exists predominantly as a hexamer, with s0(20,w) = 9.4 S and Mz approximately 600,000. In either case, the oligomers dissociate reversibly. In order to determine whether the glycosaminoglycan chains or the core protein was responsible for self-association, sedimentation velocity and sedimentation equilibrium studies were conducted on the isolated components. Dermatan sulfate chains prepared from biglycan, examined in both denaturing and physiologic solvents, show no significant difference in molecular weight (Mz approximately 22,000), whether or not the solvents contain Zn2+. However, biglycan core protein strongly self-associated in physiologic solvents. Thus, the self-association of biglycan appears to be mediated by the core protein and not by its glycosaminoglycan chains.

Interaction of heparin cofactor II with biglycan and decorin.

Two small interstitial dermatan sulfate-containing proteoglycans, biglycan and decorin, are present in extracellular matrices of skin, tendon, ligament, and cartilage. We investigated the effects of biglycan and decorin on the inhibition of alpha-thrombin by the serine proteinase inhibitor heparin cofactor II. In solution, heparin cofactor II inhibition of thrombin is accelerated by intact biglycan or decorin and by the dermatan sulfate-containing glycosaminoglycan (GAG) chains prepared from the proteoglycans, while core protein from cartilage biglycan had no effect. L-Iduronic acid-rich skin decorin and GAG chains had a greater accelerating effect than proteoglycan and GAG chains from cartilage that had lower L-iduronic acid content. Treatment of skin decorin and GAG chains with chondroitinase ABC totally eliminated the ability of these compounds to accelerate thrombin inhibition by heparin cofactor II suggesting that dermatan sulfate was responsible for this action. Both biglycan and decorin bound to type V collagen in a saturable and specific manner. Biglycan, decorin, and core protein from biglycan competed for decorin binding to the type V collagen, while only the intact proteoglycans competed for biglycan binding. When bound to type V collagen, both biglycan and decorin accelerated the heparin cofactor II/thrombin inhibition reaction as efficiently as the proteoglycans in solution. Our results demonstrate that heparin cofactor II in the presence of biglycan or decorin bound to type V collagen provides a "thromboresistant surface," further suggesting a physiological function for these proteins in regulating the extravascular activities of thrombin.

Characterization of proteoglycan-reactive T cell lines and hybridomas from mice with proteoglycan-induced arthritis.

Hyperimmunization with chondroitin sulfate-depleted fetal human cartilage proteoglycan (HFPG) leads to the development of peripheral arthritis and spondylitis in BALB/c mice. Chondroitin-sulfate-depleted adult human cartilage proteoglycan (HAPG) is much less effective at inducing arthritis. These observations suggest age differences in the presence of arthritogenic proteoglycan (PG) epitopes. Earlier studies from this laboratory have indicated an important role for PG-reactive T cells in the pathogenesis of this arthritis model. To investigate further the cellular immunity to PG in mice, two T cell lines, JY.A and JY.D, and two T cell hybridomas, TH5 and TH14, were isolated from mice with PG-induced arthritis and characterized. Two patterns of reactivity to PG emerged from the analysis of these T cells. One pattern, as demonstrated by the T cell line JY.D and the two T cell hybridomas, TH5 and TH14, was characterized by reactivity to HFPG, HAPG, chondroitin sulfate-depleted bovine cartilage PG, the G1 domain (hyaluronate binding region) of bovine cartilage PG and bovine link protein. The epitope(s) recognized by these T cells appear to be part of the homologous regions shared between the G1 domain and the link protein. The second pattern of reactivity, as demonstrated by the T cell line JY.A, was characterized by reactivity to HFPG but not to HAPG or the other PG Ag or bovine link protein. All the T cell lines and hybridomas had a CD4+, CD8- phenotype, possibly belonged to the TH1 subset (IL-2+, IL-4-), and were MHC class II restricted. These studies indicate that HFPG has T cell epitopes in common with HAPG (such as in the G1 domain) and different than those in HAPG. The significance of this data in terms of PG structure, changes with age, and induction of arthritis remains to be established.

Binding of the proteoglycan decorin to collagen type VI.

We have examined the interactions between the small dermatan sulfate proteoglycan decorin and collagen types I-VI using solid phase binding assays. The results of these studies showed that 125I-decorin bound most efficiently to collagen type VI in a time- and concentration-dependent manner. Furthermore, this interaction was specific and of moderately high affinity (Kd approximately 3 x 10(-7) M). Binding of decorin to collagen type VI appears to involve the decorin core protein rather than the glycosaminoglycan side chains, since the isolated core protein as well as a recombinant fusion protein containing a major segment (65%) of the human decorin core protein inhibited binding of 125I-decorin to collagen type VI. Other related proteoglycans and their respective core proteins also inhibited the binding of 125I-decorin to collagen type VI, whereas unrelated proteins and isolated glycosaminoglycan chains were without effect. In addition to decorin, collagen type II was also shown to bind to immobilized collagen type VI. Both interactions were effectively inhibited by preincubation of the immobilized collagen VI with decorin or collagen type II. These results suggested that the collagen type VI molecule has binding sites for collagen type II and decorin which are located in close proximity on the collagen type VI molecule. Possible functional roles of these interactions are discussed.

The predominant form of secreted colony stimulating factor-1 is a proteoglycan.

Colony stimulating factor-1 (CSF-1) is a homodimeric glycoprotein that humorally regulates the proliferation and differentiation of mononuclear phagocytic cells and locally regulates cells of the female reproductive tract. Alternative splicing of the human CSF-1 mRNA leads to alternative expression of the CSF-1 homodimer as a secreted glycoprotein or as a membrane-spanning molecule with cell surface biological activity. In the present study, analysis of immunoaffinity-purified CSF-1 from mouse L929 cell medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that CSF-1 is predominantly secreted as highly sulfated species of 375- and 250-kDa with a smaller amount of a 100-kDa species. Analysis by gel filtration in 4 M guanidine HCI buffer, indicated that, in contrast to the 100-kDa species, the highly sulfated species exhibit anomalously high molecular weights and self-association on SDS-PAGE similar to the dermatan sulfate proteoglycan, biglycan. The three predominant CSF-1 species were shown to be an 80-kDa homodimer, an 80-kDa/50-kDa heterodimer, and a 50-kDa homodimer. The 80-kDa subunit contained a single 18-kDa chondroitin sulfate chain that was absent from the 50-kDa subunit. Furthermore, treatment of the 80- and 50-kDa subunits, synthesized in the presence of tunicamycin, with chondroitinase ABC, neuraminidase, and endo-alpha-N-acetyl galactosaminidase reduced their apparent molecular masses to 60 and 25 kDa, respectively. These results are consistent with intracellular proteolytic cleavage of the 80-kDa chondroitin sulfate containing subunits from the membrane spanning CSF-1 precursor at a point carboxyl-terminal to the single consensus sequence for glycosaminoglycan addition and cleavage of the 50-kDa glycoprotein subunit at a position aminoterminal to this site. The predominance of the proteoglycan form of secreted CSF-1, which represents only 3-4% of the total trichloroacetic acid-precipitable counts released from 35SO4(2-)-labeled L cells, has important implications for regulation by this growth factor.

Extracellular matrix adhesion-promoting activities of a dermatan sulfate proteoglycan-associated protein (22K) from bovine fetal skin.

A 22 x 10(3) Mr protein (abbreviated 22K) that copurifies with dermatan sulfate proteoglycans (DS-PGs) following the biochemical fractionation of bovine fetal skin has been evaluated for adhesion-promoting activity in vitro using Balb/c 3T3 cells, as well as bovine and human dermal fibroblasts. Substrata coated with 22K protein promote attachment of a subset of 3T3 and dermal fibroblasts that respond to plasma fibronectin (pFN) substrata. Cells on 22K protein display partial cytoplasmic spreading, comparable to that of cells adhering to cell-binding fragments of pFN. Adhesion activity of 22K is not due to contamination with known adhesive proteins of dermal matrices and is not dermal cell type-specific, since two classes of neuronal cells also respond effectively to 22K substrata. DS-PGs from cartilage or skin completely inhibit 22K adhesion activity when the PGs are adsorbed to 22K substrata under conditions prohibiting PGs from binding to substrata directly. Cartilage chondroitin/keratan sulfate proteoglycan at much higher concentrations is only partially inhibitory. Inhibition by DS-PGs is mediated by DS chains binding to 22K. Properties of the cell surface 'receptor' for 22K protein were tested by several approaches. It is not cell surface DS-PG, since: (1) cells unable to produce this proteoglycan class also responded; (2) cells treated with chondroitinase ABC responded equally well; and (3) substrata of proteoglycan-binding platelet factor-4 generated responses from cells that were quantitatively and qualitatively different. A synthetic peptide in the medium containing the Arg-Gly-Asp-Ser (RGDS) sequence completely inhibited responses to 22K substrata. This observation, coupled with sequencing data of 22K protein revealing an Arg-Gly-Ala-Thr sequence at residues 151-154, suggest that 22K protein mediates adhesion by cell surface integrin binding. Therefore, this newly discovered matrix protein from skin may serve as a communication link between the dermal fibroblast cell surface and its extracellular matrix environment.

Proteoglycans of bovine articular cartilage. The effects of divalent cations on the biochemical properties of link protein.

In cartilage proteoglycan aggregates, link protein stabilizes the binding of proteoglycan monomers to hyaluronate by binding simultaneously to hyaluronate and to the G1 globular domain of proteoglycan monomer core protein. Studies reported here involving metal chelate affinity chromatography demonstrate that link protein is a metalloprotein that binds Zn2+, Ni2+, and Co2+. Zn2+ and Ni2+ decrease the solubility of link protein and result in its precipitation. However, link protein is readily soluble and functional in low ionic strength solvents from which divalent cations have been removed with Chelex 100. These observations make it possible to study the biochemical properties of link protein in low ionic strength, physiologic solvents. Studies were carried out to define the oligomeric state of link protein alone in physiologic solvents, and the transformation in oligomeric state that occurs when link protein binds hyaluronate. Sedimentation equilibrium studies demonstrate that in 0.15 M NaCl, 5 mM EDTA, 50 mM Tris, pH 7, link protein exists as a monomer-hexamer equilibrium controlled by a formation constant of 2 x 10(27) M-5, yielding a delta G' of -36 kcal/mol for the formation of the hexamer from six monomers. On binding hyaluronate oligosaccharides (HA10 or HA12), link protein dissociates to dimer. Link protein hexamer is rendered insoluble by Zn2+. Greater than 90% of the protein is precipitated by 2 mol of Zn2+/mol of link protein monomer. The binding of hyaluronate oligosaccharide by link protein strongly inhibits the precipitation of link protein by Zn2+. The link protein/hyaluronate oligosaccharide complex is completely soluble in the presence of 2 mol of Zn2+/mol of link protein. At higher molar ratios of Zn2+/link protein, the inhibitory effect of hyaluronate oligosaccharide on the precipitation of link protein is gradually overcome. Hyaluronate oligosaccharide is not dissociated from link protein by Zn2+. Hyaluronate remains bound to the link protein which is precipitated by Zn2+, or to the link protein which binds to Zn2(+)-charged iminodiacetate-Sepharose columns. Hyaluronate oligosaccharides and Zn2+ bind to different sites on link protein.

Viscoelastic and rheological properties of concentrated solutions of proteoglycan subunit and proteoglycan aggregate.

The oscillatory and steady shear rheological properties of concentrated solutions of proteoglycan subunit (PGS) and aggregate (PGA) from bovine articular cartilage have been studied using a Rheometrics fluids spectrometer. At comparable concentrations in the physiological range tan delta increases from 0.5 to 1.0 for PGA as the oscillation frequency (omega) increases from 10(-1) to 10(2) rads/s, compared to a decrease from 40 to 5 for PGS. Thus PGA solutions exhibit predominantly elastic response whereas those of PGS exhibit primarily viscous behavior. PGA solutions show pronounced shear-thinning behavior at all shear rates (gamma) in the range 10(-2) less than gamma (s-1) less than 10(2), whereas PGS solutions exhibit predominantly Newtonian flow. For PGA, the small-strain complex viscosity eta* (omega) is substantially smaller than the steady-flow viscosity eta(gamma) at comparable values of omega and gamma. These observations indicate that the presence of proteoglycan aggregates leads to formation of a transient or weak-gel network. Since aggregation leads to a large increase in molecular hydrodynamic volume and hence in the relaxation times for macromolecular rotation, it appears that role of aggregate formation is to shift the linear viscoelastic response from the terminal viscous flow into the plateau elastomeric regime of relaxational behavior. Normal or pathological changes that produce a decrease in aggregation will result in a loss of elastomeric behavior of the proteoglycan matrix.

Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor.

Confluent cultures of human endothelial cells deposit into extracellular matrix (ECM) distinct heparan sulfate proteoglycans (HSPG) which modulate acidic fibroblast growth factor's (aFGF) ability to stimulate human endothelial cell mitogenic capacity. Extracellular matrix 35S-HSPG were isolated from cultures metabolically labelled with Na235SO4 by DEAE-Sepharose, Sepharose CL-4B, and aFGF-Affi-Gel 15 column chromatography and identified by resistance to chondroitinase ABC and sensitivity to nitrous acid. Fifty to sixty percent of the 35S-HSPG deposited into ECM do not bind aFGF. The bound 35S-HSGP (40-50% of the total counts applied) eluted from the aFGF-Affi-Gel column after the addition of buffer containing 2 M NaCl. aFGF-binding and aFGF-nonbinding 35S-HSPG were individually pooled and further purified by Sepharose CL-4B column chromatography. 35S-HSPG which bind aFGF, designated HSPGP, were 100-fold superior to heparin in augmenting the mitogenic efficacy of aFGF in sparse proliferating cultures. In contrast, however, 35S-HSPG, which did not bind aFGF, designated HSPG1, inhibited aFGF-stimulated proliferation in both sparse and subconfluent endothelial cell cultures. The majority of the biological activity of both aFGF-potentiating HSPGP and aFGF-inhibitory HSPG1 was contained in the glycosaminoglycan chains released by alkaline borohydride treatment of intact HSPGP or HSPG1, respectively. 3H-Core protein derived from HSPGP or HSPG1 contained only minor biological activity. The ability of heparitinase or heparinase (Flavobacterium heparinum) to abolish biological activity differed, depending upon the HSPG tested, also suggested that these are two distinct HSPGs.

The primary structure of the core protein of the small, leucine-rich proteoglycan (PG I) from bovine articular cartilage.

Two forms of small, interstitial proteoglycans have been isolated from bovine articular cartilage and have different core proteins, based on NH2-terminal analysis and peptide mapping (Choi, H. U., Johnson, T. L., Pal, S., Tang, L-H., Rosenberg, L. C., and Neame, P. J. (1989) J. Biol. Chem. 264, 2876-2884). These proteoglycans have been called PG I and PG II. Since they were first described, they have also been called "biglycan" (PG I), "decorin," and "DS-PG" (PG II). This report describes the primary structure of PG I from bovine articular cartilage. The protein core consists of 331 amino acids with a molecular mass of 37,280 Da. The amino acid sequence shows 55% identity to the cDNA-derived sequence of PG II from bovine bone. There are four discrete domains in the amino acid sequence. Domain 1, at the NH2 terminus (approximately 23 amino acids), contains two sites of attachment of dermatan sulfate, both of which match the consensus sequence of Asp/Glu-X-X-Ser-Gly-hydrophobic. Neither of these sites is substituted to 100% with glycosaminoglycan in native PG I. Domain 2, near the NH2 terminus and containing approximately 28 amino acids, has a cysteine pattern similar to a domain near the COOH terminus of mouse metallothionein and contains at least one disulfide bond (between the first and fourth cysteine residues). The majority of the core protein of PG I (domain 3) is a leucine-rich domain containing ten repeating units (approximately 231 amino acids). Patthy [1987) J. Mol. Biol. 198, 567-577) has shown that for PG II, the majority of domain 3 shows considerable similarity to leucine-rich alpha 2-glycoprotein (LRG) from serum. Domain 2 of PG I or PG II also has an analog in LRG, in that it has two cysteines in a similar place. The major motif in the PG I described here, in PG II and in LRG, is a series of leucine-rich repeats. PG I and PG II both contain 10 leucine-rich repeats which are 14 amino acids long and which are somewhat irregularly spaced, while LRG contains 9 leucine-rich repeats spaced 10 amino acids apart. Other proteins which contain leucine repeats are the platelet glycoprotein Ib, which is involved in platelet adherence to subendothelium (eight repeats in the alpha chain and two in the beta chain), the protein encoded by the Toll gene (involved in lateral and ventral spatial organization in Drosophila) and chaoptin (a protein involved in Drosophila photoreceptor morphogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)

The isolation and primary structure of a 22-kDa extracellular matrix protein from bovine skin.

The primary structure of a 22-kDa protein which was isolated during the purification of bovine skin dermatan sulfate proteoglycan is described. The uronate-rich fraction from DEAE-Sepharose chromatography of a 7.8 M urea extract of bovine fetal skin was subjected to gel filtration on Sepharose CL-6B in 4 M guanidine HCl. A prominent component of mass 22 kDa was separated from the proteoglycan and further purified on octyl-Sepharose. The primary structure of this component was determined and found to contain three repeat regions. Each of the three sections contains a similar pattern of looped disulfide bonds. A six-amino acid consensus sequence, Asp-Arg-Glx-Trp-Asn/Gln/Lys-Phe/Tyr, is found in each loop. This domain may be involved in associations of the molecule with other extracellular matrix components.

Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography.

Two forms of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from both bovine fetal skin and calf articular cartilage and characterized. The proteoglycans were isolated using either (a) molecular sieve chromatography under conditions where DS-PGI selectively self-associates or (b) chromatography on octyl-Sepharose, which separates DS-PGI from DS-PGII based on differences in the hydrophobic properties of their core proteins. The NH2-terminal amino acid sequence of DS-PGI from skin and cartilage is identical. The NH2-terminal amino acid sequence of DS-PGII from skin and cartilage is identical. However, the amino acid sequence data and tryptic peptide maps demonstrate that the core proteins of DS-PGI and DS-PGII differ in primary structure. In DS-PGI from bovine fetal skin, 81-84% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4) disaccharide repeating units. In DS-PGI from calf articular cartilage, only 25-29% of the glycosaminoglycan was composed of IdoA-GalNAc(SO4). In DS-PGII from bovine fetal skin, 85-93% of the glycosaminoglycan was IdoA-GalNAc(SO4), whereas in DS-PGII from calf articular cartilage, only 40-44% of the glycosaminoglycan was IdoA-GalNAc(SO4). Thus, analogous proteoglycans from two different tissues, such as DS-PGI from skin and cartilage, possess a core protein with the same primary structure, yet contain glycosaminoglycan chains which differ greatly in iduronic acid content. These differences in the composition of the glycosaminoglycan chains must be determined by tissue-specific mechanisms which regulate the degree of epimerization of GlcA-GalNAc(SO4) into IdoA-GalNAc(SO4) and not by the primary structure of the core protein.

Proteoglycans of bovine articular cartilage. Studies of the direct interaction of link protein with hyaluronate in the absence of proteoglycan monomer.

When link protein binds to hyaluronate in the absence of proteoglycan monomer a high molecular weight complex is formed. Two assay procedures have been developed to examine the formation of the complex and the rate and stoichiometry of binding of link protein to hyaluronate in the complex. In the first, the complex is isolated by differential centrifugation, and the stoichiometry of binding of link protein to hyaluronate in the sedimented complex is determined. In the second assay, which involves turbidimetry, the rate of complex formation (delta A420/min) is determined, and the amount of complex formed is determined in terms of the maximum turbidity (A420,max) attained. The effects of temperature, pH, initial total solute concentration, and the ratio by weight of link protein to hyaluronate on the amount of complex formed and on the rate of complex formation were examined. There is a linear correlation between the amount of complex formed as determined by turbidity and by differential centrifugation. Using these assays, we examined the specificity of the binding of link protein to hyaluronate and the capacity of hyaluronate oligosaccharides to competitively inhibit the binding of link protein to hyaluronate. Hyaluronate decasaccharide is the oligosaccharide of minimum size that strongly inhibits the binding of link protein to hyaluronate. Proteoglycan monomers dissociate from hyaluronate as the pH is decreased from pH 7 to pH 5. Turbidimetric studies show that the rate of binding of link protein to hyaluronate increases with decreasing pH. The binding affinity of proteoglycan monomers for hyaluronate is decreased at pH 5, whereas the binding affinity of link protein for hyaluronate is not. This difference in the effect of pH on the stability of binding of link protein to hyaluronate, compared with proteoglycan monomer, explains in part the capacity of link protein to stabilize the binding of proteoglycan monomer to hyaluronate at pH 5.

Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans.

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain.

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/KS-PGs) 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 absorbed 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 3T3 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological roles of dermatan sulphate proteoglycans.

Dermatan sulphate-containing proteoglycans (DS-PGs) are widely distributed in the extracellular matrix of skin, sclera, tendon, cartilage and a variety of other connective tissues. Two species of dermatan sulphate proteoglycans, called DS-PGI and DS-PGII, have recently been isolated from mature bovine articular cartilages. In their monomeric forms, both DS-PGI and DS-PGII are polydisperse, have relative molecular masses (Mr) ranging from 80K to 140K, and possess protein cores with apparent Mr values of approximately 45K. DS-PGI readily self-associates whereas DS-PGII does not. Polyclonal and monoclonal antibodies against DS-PGII do not react with DS-PGI. DS-PGI and DS-PGII appear to possess different core proteins and represent two different species of dermatan sulphate proteoglycans. DS-PGs have dramatic effects on the biological functions of cells. For example, they inhibit the capacity of fibroblasts to adhere to a fibronectin substratum. BALB/c 3T3 cells were labelled with [3H]thymidine and plated onto dishes coated with plasma fibronectin, plasma fibronectin plus chondroitin sulphate proteoglycan (CS-PG, cartilage-specific proteoglycan monomer), or plasma fibronectin plus DS-PGs. In the absence of proteoglycan, approximately 55% of the cells were attached at 1 h. In the presence of CS-PG, cell attachment was slightly decreased. In the presence of DS-PGs, the adhesion of the fibroblasts to fibronectin was essentially abolished. Similar results were obtained if a plasma fibronectin substratum was preadsorbed with the DS-PGs and the DS-PGs were left in the attachment medium.

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography.

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.

Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages.

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.